VWF suppresses FVIII-specific recall response in vitro and in vivo. (A) Model of FVIII-specific recall response in vitro. Splenocytes obtained from immunized F8^−/− mice were stimulated with rhFVIII in the presence or absence of VWF for 6 days. FVIII-specific ASCs were detected by enzyme-linked immunospot assay. Three independent experiments done in duplicates are shown. Median and 95% confidence intervals as well as individual biological points are shown. One-way ANOVA on ranks (Kruskal-Wallis test) with Dunn multiple comparisons test was performed. (B) FVIII-specific recall response in vivo. Adoptive transfer of primed splenocytes into naïve F8^−/− mice, followed by exposure to rhFVIII with or without VWF. The anti-FVIII IgG titer was quantified at indicated time points by ELISA. Individual data points for each mouse are shown (jittered by a factor 0.8 to prevent over plotting). The curves and shaded areas are mean and 95% confidence intervals of polynomial regression. Groups were compared by 2-way ANOVA with Tukey multiple comparisons test. Statistical significance: ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.