Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice.

Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti-factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16-disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T- and B-cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti-FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII-treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII-treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro-environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain-dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.

Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells.

BACKGROUND: The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A. OBJECTIVE: We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells. METHODS: The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII-iDCs) in the presence or absence of the NFkappaB pathway blocking compound Andrographolide (Andro-cFVIII-iDCs). Three weekly intravenous infusions of one million cFVIII pulsed-iDCs were administered to a group of five hemophilic Balb/c mice. Anti-FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII. RESULTS: We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co-cultured T cells. Following repeated infusion of the cFVIII-iDCs and Andro-cFVIII-iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long-term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T-cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII-iDC infusion and the elaboration of the immunosuppressive cytokines IL-10 and TGF-beta following andrographolide-treated cFVIII-iDCs. CONCLUSIONS: This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.