Flt3L combined with rapamycin prevents antigen-specific immune response in gene and protein therapy models. (A) Experimental timeline of C57BL/6 mice that were treated with combinations of Flt3L/OVA323-339, OVA323-339/rapamycin, or Flt3L/OVA323-339/rapamycin (3 times per week for 3 weeks). Treatment was followed by challenge with scAAV1-CMV-OVA, delivered by intramuscular injection. (B) OVA323-339-specific CD8+ responses from blood were enumerated at 2 and 4 weeks postvector injection. Representative dot plots of OVA323-339-specific tetramer labeling are shown for 2 treatment conditions: Flt3L/OVA323-339 and Flt3L/OVA323-339/rapamycin. (C) Experimental timeline. Male F8e16−/−BALB/c mice were treated 3 times per week IV with combinations of Flt3L/rapamycin/low-dose FVIII (0.3 IU/mL), or rapamycin/FVIII, or Flt3L/FVIII for 4 weeks (using 0.3 IU FVIII per dose per mouse). Mice were subsequently challenged with FVIII replacement therapy (1 IU IV, 1 time per week for 4 more weeks). Control mice received the FVIII challenge only (ie, without prior immune tolerance regimen). Blood was collected on weeks 7 and 9. (D) Antibody titers against FVIII were determined by Bethesda assay and by FVIII-specific IgG1 enzyme-linked immunosorbent assay. (E) Timeline for male F8e16−/−BALB/c mice that were treated three times per week IV with Flt3L/low-dose FVIII (0.3 IU/mL) for 4 weeks. Mice were subsequently challenged with FVIII replacement therapy (1 IU IV, 1 time per week for 4 more weeks). (F) Mice pretreated with the Flt3L/FVIII combination developed high-titer inhibitor inhibitors (“Pre’), which further increased after FVIII challenge (“Post”). IgG1 responses were similarly elevated.
