Immunized E16 recipients of B-cell blasts transduced with C2-IgG/MSCV and A2-IgG/MSCV are hyporesponsive to fVIII and its C2 or A2 domain. Recipient E16 mice were primed by 4 weekly injections of human fVIII. LPS blasts were transduced with the indicated retroviral vectors, and then used as a source of B cells for tolerance induction on day 42. Mice were then boosted with 2 μg of human fVIII on day 49 and the animals euthanized 7 days later. T-cell proliferation was measured against C2 (A), A2 (B), and fVIII (C) as described in Figure 2. (A-C) ♦ indicates control SAG-Ig; □, C2-IgG plus A2-IgG. Total serum IgG anti-C2 (D) and anti-fVIII (E) antibody titers were determined by ELISA on days 28 and 56. One of 2 similar experiments is shown. Data represent the mean plus or minus SEM. ^*P < .05 or ^**P < .01 versus mock SAG-Ig control. Factor VIIII inhibitor titers are shown in panel F. ^*P < .05, ^**P < .01, or ^***P < .001 versus mock control or before gene therapy on day 28.