Splenocytes isolated from tolerized mice can adoptively transfer protection against anti-cFVIII inhibitor development to naive mice. (A) One million CD4⁺ splenocytes from tolerized mice that received Lenti-HCR/hAAT-cFVIII as neonates (cFVIII-Tolerized, n = 9) or mice that did not receive gene transfer and were not challenged with recombinant FVIII (Naive, n = 4) were injected intravenously into naive syngeneic sex-matched recipient hemophilia A mice. Mice that did not receive adoptive transfer of cells (No Cells, n = 4) were used as a control for inhibitor development. All recipient mice were subsequently challenged within 48 hours after cell transfer with 3 consecutive daily intravenous cFVIII injections (80 U/kg per day). Anti-FVIII inhibitor development was followed in the recipients for 5 weeks postadoptive transfer. (B-C) Splenocytes from hemophilia A mice that had received Lenti-HCR/hAAT-cFVIII as neonates (cFVIII-Tolerized, n = 5), mice that did not receive neonatal gene transfer or intravenous infusion of recombinant cFVIII (Naive, n = 5), and mice that were inhibitor positive after receiving subcutaneous infusion of Lenti-HCR/hAAT-cFVIII (Inhibitor, n = 5). One million CD4⁺CD25⁻ (B) or CD4⁺CD25⁺ (C) were infused into naive syngeneic sex-matched recipient hemophilia A mice via the tail vein. Mice that did not receive adoptive transfer of cells (No Cells, n = 5) were used as a control for inhibitor development.