Reduction in Factor VIII Inhibitors Mediated through Tolerogenic Antigen Presentation by immature Dendritic Cells.

Abstract Inhibitory antibodies are a major complication of factor VIII (FVIII) treatment in patients with hemophilia A. Hemophilic patients with inhibitors face major challenges in terms of their bleeding treatments and the cost of these therapies. The use of tolerogenic immunotherapy prior to protein replacement therapy represents a novel strategy to prevent the initiation of an immune response directed against FVIII. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a central role in initiating and directing T−cells towards either immunity or tolerance. We isolated myeloid immature dendritic cells (iDCs) from murine bone marrow and pulsed the cells with FVIII protein or the C2 domain of FVIII (FVIII−iDCs/C2−iDCs) under a non−inflammatory environment and in the presence or absence of anti−inflammatory and immunosuppressive agents such as dexamethasone (Dex) and andrographolide (Andro). One million pulsed−iDCs were infused into a group of five hemophilic Balb/c mice on a weekly basis for three weeks through tail vein injection (iv). Anti−FVIII antibody levels were monitored by functional Bethesda assay after a subsequent challenge with four weekly IV FVIII (2 IU/ml) or C2 (125 ng) injections. Flow cytometry assessment of DC maturation markers indicated that the DCs retained their immature state after pulsing with FVIII or the C2 domain in the presence of Andro or Dex. In vitro proliferation and cytokine release studies using naïve hemophilic mouse CD−4+ T cells and FVIII−iDCs or C2−iDCs in the presence/absence of Dex or Andro showed that the pulsed−iDCs are tolerogenic and result in a marked reduction in the secretion of inflammatory cytokines. We then studied the tolerogenic potential of FVIII−iDCs and C2−iDCs in vivo. We infused pulsed−iDCs that were cultured in the presence or absence of Dex/Andro into naïve hemophilic mice. We observed a 20% and 70% reduction in the levels of FVIII inhibitors in mice that received FVIII−iDCs or C2−iDCs respectively after FVIII or C2 challenges in comparison to control mice that only received FVIII or C2 challenges. Mice that received FVIII−iDCs were re−challenged with 2 IU of FVIII 10 weeks after the last of the four consecutive FVIII challenges. Our results indicate that these mice still showed a 25% reduction in the levels of FVIII inhibitors after the re−challenge suggesting that iDCs were able to induce a tolerogenic memory response against FVIII. The in vivo studies for FVIII pulsed iDCs in the presence of Andro are still in progress. In aggregate, these studies indicate that the administration of immature DCs pulsed with FVIII or the C2 domain of FVIII can significantly reduce the immune response to FVIII following subsequent IV challenges. However, this immunotherapeutic strategy does not completely abolish the anti−FVIII response, suggesting that additional supplementary approaches will be necessary to prevent the development of this treatment−related complication.

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