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

Abstract

Summary. An HLA-DRA-DRB1*0101-restricted Tcell 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 subjectsÕ 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][2195][2196][2197][2198][2199][2200][2201][2202][2203][2204][2205][2206][2207][2208][2209][2210][2211][2212][2213] . A haemophilic cousinÕs HLA-DRA-DRB1*1104-restricted response to FVIII [2202][2203][2204][2205][2206][2207][2208][2209][2210][2211][2212][2213][2214][2215][2216][2217][2218][2219][2220][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.

T‐cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

“…Our criterion for positive staining was designated as the mean background staining plus three times the standard error of the mean: 1.53% for FVIII 381–400 and 0.46% for all other specificities. The latter is consistent with the cut‐off used in previous published studies [17,18,30–33].…”

T‐cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

“…Our criterion for positive staining was designated as the mean background staining plus three times the standard error of the mean: 1.53% for FVIII 381–400 and 0.46% for all other specificities. The latter is consistent with the cut‐off used in previous published studies [17,18,30–33].…”

HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

“…Both donors that present this peptide in our study are DRB1*0101 as well. Two other C2 peptides identified in this study were found previously to induce T-cell proliferation (35,37). The fact that most peptides found were donor-specific suggests that these peptides are presented in a HLA-specific context.…”

Specificity of the T Cell Response to Protein Biopharmaceuticals

“…Both the epitope with the R2150H mutation and the non-mutated counterpart bind with high affinity to multiple HLA-DR molecules, the mutation supposedly being in tight contact with the TCR ( 38 ). The epitope with the A2201P mutation ( 39 , 105 ) was the same as that found in severe hemophilia A patients ( 41 ). In contrast to the mutated regions, the remaining parts of FVIII did not give rise to specific T cells in the mild/moderate hemophilia A patients and appeared to be tolerated ( Figure 3 c).…”

“…In FVIII-deficient HLA-DR15-transgenic mice, which are designed to mimic severe hemophilia A, the T cell response is supported by 8 dominant epitopes, which exhibit a good affinity for different HLA-DR molecules. T cell epitopes identified in FVIII ( 36 , 38 , 39 , 106 ) are some of the presented peptides ( 108 ) but all the presented FVIII peptides did not elicit a T cell response as already observed for therapeutic mAbs ( 53 , 72 ). The T cell response in severe hemophilia A patients may appear to focus on only one high-avidity epitope, when assessed with HLA-Class II tetramers ( 41 ), which is at variance with studies using T cell proliferation assays suggesting a much broader T cell response to FVIII ( 34 ).…”

T‐cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

“…Our criterion for positive staining was designated as the mean background staining plus three times the standard error of the mean: 1.53% for FVIII 381–400 and 0.46% for all other specificities. The latter is consistent with the cut‐off used in previous published studies [17,18,30–33].…”

HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

“…Both donors that present this peptide in our study are DRB1*0101 as well. Two other C2 peptides identified in this study were found previously to induce T-cell proliferation (35,37). The fact that most peptides found were donor-specific suggests that these peptides are presented in a HLA-specific context.…”

Specificity of the T Cell Response to Protein Biopharmaceuticals

“…Both the epitope with the R2150H mutation and the non-mutated counterpart bind with high affinity to multiple HLA-DR molecules, the mutation supposedly being in tight contact with the TCR ( 38 ). The epitope with the A2201P mutation ( 39 , 105 ) was the same as that found in severe hemophilia A patients ( 41 ). In contrast to the mutated regions, the remaining parts of FVIII did not give rise to specific T cells in the mild/moderate hemophilia A patients and appeared to be tolerated ( Figure 3 c).…”

“…In FVIII-deficient HLA-DR15-transgenic mice, which are designed to mimic severe hemophilia A, the T cell response is supported by 8 dominant epitopes, which exhibit a good affinity for different HLA-DR molecules. T cell epitopes identified in FVIII ( 36 , 38 , 39 , 106 ) are some of the presented peptides ( 108 ) but all the presented FVIII peptides did not elicit a T cell response as already observed for therapeutic mAbs ( 53 , 72 ). The T cell response in severe hemophilia A patients may appear to focus on only one high-avidity epitope, when assessed with HLA-Class II tetramers ( 41 ), which is at variance with studies using T cell proliferation assays suggesting a much broader T cell response to FVIII ( 34 ).…”

T‐cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

“…Our criterion for positive staining was designated as the mean background staining plus three times the standard error of the mean: 1.53% for FVIII 381–400 and 0.46% for all other specificities. The latter is consistent with the cut‐off used in previous published studies [17,18,30–33].…”

HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

“…Both donors that present this peptide in our study are DRB1*0101 as well. Two other C2 peptides identified in this study were found previously to induce T-cell proliferation (35,37). The fact that most peptides found were donor-specific suggests that these peptides are presented in a HLA-specific context.…”

Specificity of the T Cell Response to Protein Biopharmaceuticals

“…Both the epitope with the R2150H mutation and the non-mutated counterpart bind with high affinity to multiple HLA-DR molecules, the mutation supposedly being in tight contact with the TCR ( 38 ). The epitope with the A2201P mutation ( 39 , 105 ) was the same as that found in severe hemophilia A patients ( 41 ). In contrast to the mutated regions, the remaining parts of FVIII did not give rise to specific T cells in the mild/moderate hemophilia A patients and appeared to be tolerated ( Figure 3 c).…”

“…In FVIII-deficient HLA-DR15-transgenic mice, which are designed to mimic severe hemophilia A, the T cell response is supported by 8 dominant epitopes, which exhibit a good affinity for different HLA-DR molecules. T cell epitopes identified in FVIII ( 36 , 38 , 39 , 106 ) are some of the presented peptides ( 108 ) but all the presented FVIII peptides did not elicit a T cell response as already observed for therapeutic mAbs ( 53 , 72 ). The T cell response in severe hemophilia A patients may appear to focus on only one high-avidity epitope, when assessed with HLA-Class II tetramers ( 41 ), which is at variance with studies using T cell proliferation assays suggesting a much broader T cell response to FVIII ( 34 ).…”