Plasma levels of IgG1 anti-hFIX 3 weeks after immunologic challenge by subcutaneous administration of 5 μg hFIX formulated in CFA in C57BL/6 mice that had received adoptive transfer of splenocytes from naive (white bars, controls) or vector-treated (hatched bars, AAV-FIX) C57BL/6 mice. Adoptive transfer was by tail vein injection 24 hours before challenge. Vector-treated mice had received hepatic gene transfer with 10¹¹ vg/animal AAV-ApoE/hAAT-hFIX vector 6 weeks before the experiment. (A) Total splenocytes (5 × 10⁷, bars 1-2), CD4⁺ T-cell–depleted splenocytes (5 × 10⁷, bar 3), CD25⁺-depleted CD4⁺ T cells (9 × 10⁶, bars 6, 8), MACS-purified CD4⁺ T cells (10⁷, bars 4-5), or CD4⁺CD25⁺ cells (1 × 10⁶, bars 7, 9) were transferred. Each bar is average antibody titer for 6 animals (± SD). *P < .05 compared with control splenocytes. **P < .01 compared with control CD4⁺ cells. ***P < .01 compared with CD4⁺CD25⁻ cells or to control CD4⁺ cells. (B) CD4⁺GITR⁺ (bars 12-13) or CD4⁺GITR⁻ splenocytes (bars 10-11) were transferred. Each bar is average antibody titer for 4 animals (± SD). *P < .05 compared with CD4⁺GITR⁻ cells or to control CD4⁺GITR⁺ cells.