Immune responses in 2bCoF9R338L-transduced rhFIX-primed recipients. Anti-FIX antibody titers in rhFIX-primed FIX^null mice were monitored monthly after receiving 2bCoF9R338L-transduced HSCs. rhFIX-primed FIX^null mice preconditioned with 6.6 Gy TBI and transplanted with untransduced HSCs were used as controls in parallel. Anti-FIX inhibitor titers were determined by Bethesda assay, and anti-FIX total IgG titers were determined by ELISA. (A) Anti-hFIX inhibitor titers in 2bCoF9R338L-transduced rhFIX-primed FIX^null recipients. (B) Anti-hFIX total IgG titers in 2bCoF9R338L-transduced rhFIX-primed FIX^null recipients. (C) The half-life of anti-FIX inhibitor disappearance time. (D) The half-life of anti-FIX total IgG disappearance time. (E) The correlation between platelet-FIX:Ag levels and half-lives of anti-FIX inhibitor disappearance time in 2bCoF9R338L-transduced rhFIX-primed recipients. (F) The correlation between platelet-FIX:C levels and half-lives of anti-FIX inhibitor disappearance time in 2bCoF9R338L-transduced rhFIX-primed recipients. (G) The correlation between platelet-FIX:Ag levels and half-lives of anti-FIX total IgG disappearance time in 2bCoF9R338L-transduced rhFIX-primed recipients. (H) The correlation between platelet-FIX:C levels and half-lives of anti-FIX total IgG disappearance time in 2bCoF9R338L-transduced rhFIX-primed recipients. (I) Immune tolerance was established in 2bCoF9R338L-transduced rhFIX-primed primary recipients. When anti-FIX inhibitor titers dropped to undetectable, animals were challenged with rhFIX at 100 U/kg per week by IV administration for a total of 4 times. One week after the second and fourth re-challenge, blood samples were collected for the inhibitor assay. (J) Comparison of the changes in inhibitor titers in primary vs secondary recipients 1 month after to before HSCT. (K) Comparison of the half-life of anti-FIX total IgG disappearance time in primary vs secondary 2bCoF9R338L-transduced recipients. (L) Immune tolerance developed in 2bCoF9R338L-transduced secondary primed FIX^null recipients. Data are presented as mean ± SD. *P < .05; **P < .01.