Memory B-cell–mediated ELISPOT assay. Splenocytes were isolated from rhF8-immunized FVIII^null mice, and CD138⁺ plasma cells were depleted. The remaining cells (CD138⁻) were used as memory B-cell pools. CD138⁻ memory B-cell pools were cultured with various doses of rhF8 with or without 1 U/mL rhVWF for 6 days to induce differentiation of memory B cells into ASCs. ASCs were determined by ELISPOT assay. Each spot represents a single ASC. (A) The rhF8 dose response on inducing differentiation of memory B cells into ASCs and anti–FVIII antibody production. (B) Representative ELISPOT images showing the impact of VWF on memory B-cell differentiation and antibody secretion. (C) ASC counts in various culture conditions. The paired Student t test was used to compare data sets. These results demonstrate that VWF can mitigate FVIII-specific differentiation of memory B cells into ASCs and anti–FVIII antibody production.