However, if FVIII is administered when anti-CD40L is no longer present, inhibitors are not suppressed. Similar results have been observed with blockade of the CD80/86–CD28 interaction by administration of CTLA-4-Ig [31]. Additional

attempts have been made to utilize FVIII-loaded tolerogenic APCs to reduce immunogenicity. These studies have loaded immature dendritic cells with FVIII ex vivo and then re-infused the cells in an attempt to mediate tolerogenic presentation of FVIII to T cells [32]. The hypothesis being tested is that T regulatory cells (Treg) as opposed to effector cells (DCs) would be generated by these immature DC interactions. A similar strategy has been LDE225 supplier recently evaluated in which FVIII-transgene-modified apoptotic fibroblasts have been infused into haemophilic mice [33]. However, despite significant reductions in the titres of FVIII antibodies in both these approaches, neither strategy resulted in FVIII tolerance. The final approach that has been used to alter antigen presentation has involved mucosal antigen exposure through oral and nasal application of clotting factor protein. Studies using mucosal exposure to the immunogenic C2 domain have shown that tolerance to C2 can be achieved through this strategy, but when the mice are exposed to

full-length FVIII, inhibitory antibodies develop [34]. Very recent preliminary evidence suggests that FIX made in transgenic Bay 11-7085 Metabolism inhibitor plants can mediate tolerance and prevent fatal anaphylactic episodes

after oral administration [35]. Studies involving non-T-cell-depleting anti-CD4 and anti-CD3 F(ab)’2 have also been conducted in haemophilic mice. In the anti-CD4 studies, FVIII inhibitors were still generated, but at lower levels than normally observed [36]. With low-dose anti-CD3 administration, transient depletion of CD4+ T cells was observed and at the same time, a doubling of CD25+ Treg cells [37]. In these experiments, after the subsequent administration of FVIII, inhibitor development was abolished in the majority of mice and when inhibitors were observed, they were of very low titre. The tolerance induced by this protocol was dependent upon CD25+ Foxp3+ Treg cells. Finally, a B-cell-targeted gene transfer approach has also been successful in mediating FVIII tolerance. In this study, B cells from haemophilic mice were transduced ex vivo with lentivectors expressing chimeric FVIII A2-IgG or C2-IgG [38]. After re-infusion of the transduced B cells, FVIII inhibitor development was abolished in naïve mice and levels of FVIII antibody were significantly reduced in mice with pre-existing antibodies. Although the tolerance mechanism responsible for these findings is likely complex, there was again evidence that CD4+ CD25+ Treg cells played a crucial role.