Depletion of splenic MZ B cells prevents development of anti-FVIII antibodies. (A) FVIII-KO mice were treated with MZ B-cell–depleting antibodies, an isotype control antibody, or saline (red arrows) beginning 4 days before initial exposures to intermediate doses of FVIII (black arrows). Plasma was collected 1 week after the fourth weekly FVIII infusion (blue arrow) and analyzed by enzyme-linked immunosorbent assay (ELISA) (B) and Bethesda assay (C) to determine total and inhibitory anti-FVIII antibody titers, respectively. MZ B-cell depletion completely ablated the primed anti-FVIII antibody response, indicating a key role for MZ B cells in initiating the naive anti-FVIII immune response. Mice receiving subsequent FVIII injections following a delay to allow MZ B-cell reconstitution developed anti-FVIII antibodies (not depicted here), indicating that MZ B-cell depletion alone was insufficient to achieve peripheral tolerance to FVIII. BU, Bethesda units. ***P < .0002, ****P < .0001, 1-way analysis of variance, post hoc Tukey test. This figure has been adapted from Figure 3A-C in the article by Zerra et al that begins on page 2559.