Fig. 5. Endocytosis of FVIII by DCs. (A) Dose-dependent endocytosis of FVIII by DCs. DCs were incubated in X-VIVO¹⁵ medium with FVIII (8, 16, 25, 40, 75, and 100 mg/ml) for 2 h at 37°C. Cells were washed extensively and analyzed by flow cytometry. (B) DCs were cultured with FVIII-FITC (40 mg/ml) for 2 h at 37°C. Cells were then fixed (with 100% ethanol for 8 min) on a glass slide and observed with a confocal microscope (LEICA SP2; Leica, Mannheim, Germany) coupled to an inverted microscope. The detection wavelength range was 500-580 nm for FITC. Gray-level images were obtained by DIC. 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 merged using Adobe Photoshop 7.0 software.

Fig. 6. Validation of the CTLD4-7-Fc and CR-FNII-CTLD1-3-Fc constructs. (A) ELISA plates were coated with varying doses of mannan. The constructs CTLD4-7-Fc (CTLD4-7) and CR-FNII-CTLD1-3-Fc (CTLD1-3) were added to the wells at 10 mg/ml. A mouse anti-human Ig Fc-specific antibody coupled to HRP was then incubated in the wells. Binding was assessed by measuring the absorbance at 492 nm after revealing with OPD substrate. (B) ELISA plates were coated with 5 mg/ml of SO4-3-b-D-GalNAc-PAA. CR-FNII-CTLD1-3-Fc construct was added to the wells in serial dilutions starting from 10 mg/ml. A peroxidase-coupled anti-human IgG (Southern Biotech) was then incubated in the wells. Binding was assessed by measuring the absorbance at 492 nm after revealing with OPD substrate.

Fig. 7. Specificity of FVIII-specific T cell activation assay. (A) DCs (10,000 per well) generated from DRB1*1501/DRB5*01 healthy blood donors were incubated either in medium alone or in presence of mannan (1 mg/ml) or anti-CD206 IgG (10 mg/ml)m followed by incubation with FVIII (10 mg/ml, 36 nM) or I²¹⁴⁴-T²¹⁶¹ peptide (1, 2, and 4 mg/ml) and D9E9. Human factor IX (36 nM, FIX, Benefix; Baxter) was used instead of FVIII as a control. Results are from one representative of three to eight independent experiments. (B) Dose-response of mannan-mediated inhibition of FVIII-specific T cell activation. DCs were preincubated with various concentrations of mannan (0, 0.0001, 0.01, 1, and 3 mg/ml), followed by coculturing in the presence of D9E9 (5,000 per well) and FVIII (5.56, 2.78 or 1.39 mg/ml). Results are from one representative of three independent experiments.

Fig. 8. Mannan does not inhibit presentation of FVIII to D9E9 by FVIII nonspecific B lymphoblastoid lines. (A) LE56, a B lymphoblastoid cell line without a FVIII-specific BCR and HLA-DR-matched to D9E9, was incubated with medium alone or mannan (1 mg/ml) for 30 min at 37°C. Cells were then cocultured with the FVIII-specific CD4+T cell clone D9E9 in presence of BDD-FVIII (4.5 mg/ml) for 20 h. Activation of D9E9 was assessed by measuring IFN-gamma in the culture supernatant by ELISA. (B) Control antigen presenting cells (APCs) for the effect of mannan, e.g., DCs and BO2C11 (a B lymphoblastoid cell line with a FVIII-specific BCR that is HLA-DR-matched to D9E9) were treated similarly, as in the case of LE56.

Fig. 9. Preincubation of FVIII (10 mg/ml) with VWF inhibited the interaction of FVIII with CTLD 4-7-Fc in a dose-dependent manner as assessed by ELISA. Error bars represent standard deviations of two independent experiments.