Reduction of Factor VIII Inhibitor Formation with F.VIII-Pulsed Tolerogenic Dendritic Cells in the Murine Hemophilia A Model.

Abstract

Inhibitor formation is a severe complication of hemophilia, occurring in up to 25% and associated with poor response to factor replacement, uncontrolled bleeding, and high morbidity. Preventing inhibitor formation is, thus, a major goal of hemophilia management. The role of dendritic cells (DC) in regulating immune response has been increasingly recognized: immature DC (imDC) induce T regulatory cells in vitro and promote Ag-specific tolerance in vivo. We, therefore, studied the role of imDC propagated from bone marrow with GM-CSF + TGFβ to prevent inhibitor formation in the hemophilia A murine model. Following tail vein injection of recombinant F.VIII (Advate, Baxter) 2.5 U (0.2 μg) on days 0, 2, and 4 in hemophilia A exon 16 KO C57Bl/6 mice, anti-VIII antibodies were detected by semi-quantitative APTT (scored 1-4), peaking on day 6. On rechallenge with F.VIII 2.5 U on days 12, 14, and 16, anti-VIII was detected, peaking on day 17. Anti-VIII production was associated with high level splenic T cell proliferation in response to F.VIII stimulation in vitro, measured by 3H-thymidine incorporation in mixed lymphocyte reaction (MLR). By contrast, there was no antibody formation in F.VIII-treated Wt C57Bl/6 mice: the latter was associated with low T cell response to F.VIII in vitro. Functionally immature DC (imDC) were propagated from the bone marrow of hemophilia A mice with GM-CSF (4ng/ml) and TGFβ (0.2ng/ml). For comparison, functionally mature dendritic cells (mDC) were propagated with GM-CSF (4ng/ml) and IL-4 (1000U/ml).The former (imDC) demonstrated deficient NF-kB binding activity in nuclear protein as detected by gel shifting assay and expressed low level of costimulatory molecules CD80, CD86; by contrast, the latter (mDC) demonstrated enhanced NF-kB binding activity and high levels of co-stimulatory molecules. Administration of 2x106 F.VIII-pulsed imDC (20U/ml x 24h) 7 days before F.VIII dosing on days 0, 2, and 4, led to reduction in inhibitor formation on day 6 (score 1.6 vs. 2.3 in control group) which was further reduced on day 8 (score 1.0 vs. 2.0 in control group). The inhibitor could not be detected on day 8 in 2 of 4 mice pretreated with F.VIII-pulsed imDC. By contrast, high levels of inhibitor were detected in mice pretreated with F.VIII-pulsed mDC (score 3.3). Rechallenge with F.VIII on day 10 in imDC-treated mice resulted in no increase in the reduced or absent anti-VIII effect on day 12. Splenic T cells (CD3+) from the imDC-pretreated mice showed lower proliferative capacity when restimulated in vitro with F.VIII, suggesting that imDC induced F.VIII unresponsiveness. These studies show that FVIII-pulsed imDC reduce the intensity of inhibitor formation, and suggest the potential role of modified DC in preventing or reducing F.VIII inhibitor formation.

Cellular and genetic therapies for haemophilia

“…Finally, new approaches for inducing immunological tolerance to the clotting factor proteins are also being explored and might play a role in reducing the risk of inhibitor development in gene transfer studies. Tolerogenic presentation of the clotting protein through either the mucosal surfaces of the nasal passages [23] or gut [24], or through presentation by immature dendritic cells [25] have all shown early promise in preclinical investigations.…”