Suppression of the Immune Response to Human FVIII with FVIII Transgene Modified Tolerogenic Dendritic Cells in Hemophilic Mice

The development of anti-factor VIII (FVIII) inhibitory antibodies is currently the most significant complication of FVIII replacement therapy in the management of patients with hemophilia A. Infusion of in vitro generated tolerogenic dendritic cells (tDCs) loaded with foreign antigen has been shown to promote durable antigen-specific tolerance in vivo through mechanisms that involve the induction of regulatory T cells. In this study we evaluated the ability of tDCs transduced with a human B domain deleted FVIII transgene-expressing foamy virus (FV) vector to modulate the immune response to human FVIII in both naïve and pre-immunized hemophilia A mice. The tDCs were generated by flow sorting the population of CD11c^lowCD45RB^high cells produced in culture of lineage negative bone marrow cells in RPMI1640/10%FBS supplemented with IL-10 and the neural peptides VIP and PACAP38. Expression of co-stimulatory molecules CD80 and CD86 and MHC Class II was negative or low on the generated tDCs and these cells remained un-activated even after stimulation with LPS or transduction by FV vectors. These tDCs produced low levels of IL-6 and TNF-α, and high level of IL-10. Furthermore, co-culture of the vector transduced tDCs with FVIII stimulated effector T cells (Teffs) resulted in decreased proliferation of Teffs and reduced secretion of IFN-γ and IL-2. In the cultures with the transduced tDCs there was also an increase in the number of apoptotic Teffs. Naïve Balb/c hemophilia A mice were treated with 2 weekly infusions of FVIII vector transduced tDCs (tDC-F8), control tDCs (tDCs-Ctrl), or no cells (Neg-Ctrl) prior to being challenged with four weekly intravenous doses of 0.2 μg rhFVIII. Following immunization the total cellularity and weights of spleens harvested from tDC-F8 mice were consistently half that of spleens from either tDC-Ctrl or Neg-Ctrl mice. Furthermore, inhibitor titers in tDC-F8 mice were 60–61% lower than either Neg-Ctrl or tDC-Ctrl mice (p < 0.05 compared to both controls). The regulatory T cell related markers FOXP3, CD25, CD103, CTLA4 and GITR were all up-regulated on splenic CD4⁺ T cells from tDC-F8 mice and the CD4⁺ T cell proliferation response to FVIII stimulation in splenocytes from tDC-F8 mice was suppressed by approximately 90%. Moreover, the rate of apoptosis in splenic T cells from tDC-F8 mice was 33% higher than splenic T cells from either Neg-Ctrl or tDC-Ctrl mice. In pre-immunized mice, treatment with 4 weekly infusions of FVIII vector transduced tDCs lowered inhibitor titers by 54% compared to no treatment controls (p < 0.05). In contrast, treatment with untransduced tDCs had no significant effect on the inhibitor titers of pre-immunized mice. Importantly, adoptive transfer of CD4⁺ T cells from tDC-8 mice produced suppression of the immune response to FVIII in subsequently immunized naïve secondary recipients.. In summary, these data indicate that FVIII vector transduced tDCs are useful in suppressing the immune response to FVIII in hemophilia A mice and suggest that regulatory T cells play a role in the induced immune modulation. More in vivo studies are in progress to confirm the durability of these effects. Future studies will also focus on isolating and characterizing the regulatory T cell populations induced by in vivo administration of transgene modified tDCs.