VWF reduces FVIII endocytosis by DCs and the consequent presentation to FVIII-specific T cells. (A) Reduction of FVIII endocytosis by DCs in the presence of VWF. FVIII-FITC (107 nM) was incubated with DCs for 2 hours at 37°C, following preincubation in medium alone or in the presence of increasing concentrations of VWF or HSA (ie, molar ratios of 1:1 to 1:130). Cells were then analyzed by flow cytometry. Results depict relative percentage of FVIII-FITC⁺ cells considering 100% when preincubation was done in medium alone. Data are the average of 6 independent experiments. Significant differences were assessed using the Mann-Whitney test. (B) Reduction of the activation of D9E9 in the presence of VWF. FVIII (20 nM) and the I²¹⁴⁴-T²¹⁶¹ peptide (2 üg/mL) were incubated with DCs (10 000 cells/well) and D9E9 (5000 cells/well) in medium alone or in the presence of increasing amounts of VWF, for 20 hours at 37°C. Activation of D9E9 was assessed by measuring the released IFN-γ by ELISA. Data are from 1 representative experiment. (C) Specificity of the protective effect of VWF on FVIII endocytosis by DCs. FVIII-FITC (107 nM) and α2M-FITC (27.8 nM), preincubated alone or with a 25-fold molar excess of VWF, were incubated with DCs for 2 hours at 37°C. Ligand-FITC⁺ cells (ie, FVIII-FITC or α2M-FITC) were analyzed as described. Average of the results obtained with cells from 2 different donors. (D) Requirement of FVIII-VWF binding for inhibition of FVIII endocytosis. FVIII (5 nM) was incubated with DCs (10 000 cells/well) and D9E9 (5000 cells/well) in medium alone or in the presence of full-length recombinant VWF (recVWF) or D′D3 deleted recombinant VWF (ΔD′D3 recVWF), for 20 hours at 37°C. Activation of D9E9 was assessed by measuring the released IFN-γ by ELISA. Statistical significance was assessed using the ANOVA Fisher PLSD test. Error bars indicate SD.