Factor VIII−pulsed dendritic cells reduce anti−factor VIII antibody formation in the hemophilia A mouse model

ObjectiveHemophilia inhibitor formation is a T-cell−dependent immune response to infused factor VIII (F.VIII). As immature dendritic cells (DCre) regulate immune response and promote tolerance, we evaluated F.VIII-pulsed DCre, propagated from bone marrow in the presence of granulocyte-macrophage colony-stimulating factor and transforming growth factor−β, in achieving F.VIII tolerance.Materials and MethodsThe effects of intravenous F.VIII-pulsed DCre in C57BL/6 hemophilia A mice were determined by total F.VIII inhibitory antibodies, T-cell proliferation, thymidine uptake, cytokine profile, and surface molecule expression.ResultsAfter tail vein injection of 2.5 U recombinant F.VIII (rF.VIII) on day 0, 2, and 4, anti-F.VIII antibody peaked on day 6, and increased further on day 17 following rF.VIII rechallenge on day 12, 14, and 16, with increased T-cell proliferative response to in vitro F.VIII. When mice were pretreated with 2 × 106 F.VIII-pulsed immature DCre (deficient nuclear factor-κB nuclear protein binding, low CD80, low CD86, high interleukin [IL]-10 phenotype) 7 days before rF.VIII challenge, anti-F.VIII was reduced on day 6 and on day 8, 0.1 ± 0.0 (Bethesda units/mL) vs control phosphate-buffered saline−treated hemophilia A mice, 2.0 ± 0.1 Bethesda units/mL, p < 0.01. Rechallenge with rF.VIII on day 12 produced no increase in anti-F.VIII antibody response. This was associated with high serum IL-10 and low IL-2 levels by enzyme-linked immunosorbent assay, and splenic T-cell hyporesponsivess to F.VIII, with IL-10 production, high FoxP3 expression by quantitative polymerase chain reaction, and T regulatory cell expansion, confirmed in ovalbumin−T-cell receptor transgenic mice.ConclusionsThese findings suggest F.VIII-pulsed DCre reduce anti-F.VIII antibody formation in hemophilia A mice by induction of regulatory T-cell−mediated hyporesponsiveness of T-helper cells to F.VIII.