Different frequencies of FVIII-specific CD4⁺ T cells in the spleen after intravenous and subcutaneous application of FVIII in E17 HLA-DRB1*1501 mice. Spleen cells were prepared from mice treated with 8 weekly doses of intravenous (B) or subcutaneous (C) FVIII and in vitro restimulated with either full-length human FVIII (B-C; 20 μg/mL) or FVIII peptides (C; 1 μg/mL per peptide) for 16 hours. The frequency of FVIII-specific or peptide-specific CD4⁺ T cells expressed as the percentage of CD4⁺ CD154⁺ T cells in relation to total CD4⁺ T cells is presented. Data are the results of a representative experiment. A total of 3 experiments with identical settings were done; all generated similar data. (A) The gating strategy for the quantification of CD4⁺ T cells that express intracellular CD154. (B) Frequencies of FVIII-specific CD4⁺ T cells (CD4⁺ T cells expressing intracellular CD154) after intravenous FVIII treatment. The results presented were obtained from mice with (intravenous responder) and without (intravenous nonresponder) anti-FVIII antibodies. Naive mice (naive) were included as a negative control. (C) Frequencies of FVIII-specific CD4⁺ T cells (CD4⁺ T cells expressing intracellular CD154) after subcutaneous FVIII treatment. Spleen cells were restimulated with 1 of the 6 following items: FVIII (FVIII), a mix of the 3 FVIII epitopes identified as dominant epitopes in E17 HLA-DRB1*1501 mice as shown in Figure 3A (peptide mix), a mix of scrambled versions of the 3 dominant epitopes (peptide control A), a mix of 3 FVIII epitopes identified as dominant epitopes in conventional E17 mice, which express the murine MHC class II molecule (peptide control B), a peptide pool spanning the C2 domain of FVIII, and one of the individual FVIII peptide regions containing dominant epitopes in E17 HLA-DRB1*1501 mice as indicated. Spleen cells restimulated with medium only (medium) were included as a negative control.