Endocytosis of FVIII by human DCs and presentation to FVIII-specific T cells. (A) Dose-dependent labeling of DCs following incubation with FVIII-FITC. Five-day-old monocyte-derived human DCs were incubated in X-VIVO¹⁵ medium containing 0 to 357 nM FVIII-FITC for 2 hours at 37°C. Following incubation, cells were washed extensively and fluorescent cells were analyzed by flow cytometry. Representative of more than 2 experiments. (B) Intracellular localization of FITC-FVIII. DCs were incubated with FVIII-FITC for 2 hours at 37°C. Cells were then fixed on a glass slide and observed with a confocal microscope equipped with a 63 ×/1.32 numerical aperture oil objective. Gray-level images were obtained by differential interference contrast. The fluorescence image was chosen in the middle of the cell, at the level of the nuclei. The fluorescence image and the corresponding gray-level image were acquired using Leica Confocal System software, and were merged using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). (C) Activation of the FVIII-specific CD4⁺ T-cell clone D9E9 by FVIII-loaded DCs. DCs were generated from blood donors with matched MHC haplotypes (thanks to J. Treton, Institut National de la Santé et de la Recherche Médicale [INSERM] Unité [U] 662, Paris, France) and were incubated at 10 000 cells/well with D9E9 (5000 cells/well) in DMEM-10% FCS-20 IU/mL rhIL-2, alone or in the presence of 0 to 36 nM FVIII for 20 hours at 37°C. Activation of D9E9 was assessed by measuring released IFN-gamma by enzyme-linked immunosorbent assay (ELISA). Data are representative of 3 independent experiments. (D) DCs (10 000 cells/well) were cultured with D9E9 (5000 cells/well) alone or in the presence of 20 nM FVIII, human recombinant factor IX (FIX, 36 nM) or a synthetic FVIII peptide (I²¹⁴⁴-T²¹⁶¹) known to activate D9E9 (2 üg/mL), for 20 hours at 37°C. Data represent the average of 7 independent experiments. Error bars indicate SD.