Immune response after intravenous and subcutaneous FVIIItreatment of E17 HLA-DRB1*1501 mice is driven by the same immunodominant CD4⁺ T-cell epitopes, which are completely different from CD4⁺ T-cell epitopes found in E17 mice expressing a murine MHC class II complex. E17 HLA-DRB1*1501 mice (E17 human MHC class II) and conventional E17 mice (E17 murine MHC class II) were immunized 4 to 8 times by intravenous or subcutaneous FVIII at weekly intervals. Spleen cells were obtained at 3 to 7 days after the last immunization and restimulated with FVIII in vitro. T-cell hybridomas were generated and characterized for their peptide specificity using a 15-mer peptide library (12 amino acids overlap) covering the sequence of full-length FVIII. Peptide specificities for all T-cell hybridoma clones could be identified, indicating that the large number of peptides per pool did not interfere with peptide recognition. (A) The number of T-cell hybridomas obtained from E17 HLA-DRB1*1501 mice that were identified for each of the indicated FVIII epitopes. A total of 118 hybridomas obtained after subcutaneous FVIII and 63 hybridomas obtained after intravenous FVIII treatment were analyzed. (B) The distribution of CD4⁺ T-cell epitopes identified in E17 HLA-DRB1*1501 mice (E17 human MHC class II) and in conventional E17 mice (E17 murine MHC class II) over the different domains (A1, a1, A2, a2, B, a3, A3, C1, and C2) of FVIII.