Treatment with monoclonal anti-CD25 prevents the tolerogenic effects of B-cell gene therapy. E16 mice were treated with PC61 anti-CD25 antibody or normal rat IgG and injected with B cells transduced with retroviral vectors expressing C2-IgG plus A2-IgG or control SAG-IgG. The efficacy of PC61 depletion was validated by flow cytometric analysis of peripheral blood leukocytes obtained from treated mice on day 9. The percentage of CD25⁺ cells is indicated in the top right quadrant. (A). Mice were challenged repeatedly with human fVIII as in Figure 2. Animals were euthanized at 5 days after final fVIII injection and measures of cellular and humoral immunity were taken as described in “Materials and methods.” T-cell responses are depicted in panels B and C (B: ♦, C2-IgG + A2-IgG; ○, SAG-Ig; C: ▵, C2-IgG + A2-IgG; ▪, SAG), and ELISA titers are shown in panels D and E. Inhibitory antibody titers are shown in panel F. ^*P < .05 or ^**P < .01 versus mock control. Error bars indicate mean ± SEM for individual mice of the cpm incorporated minus background.