PB1049 - Immune modulatory function of desialylated platelets containing FVIII in FVIII immune responses in hemophilia A mice

Background: The development of anti-FVIII inhibitory antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A (HA) patients. Our previous studies have demonstrated that platelet-targeted FVIII gene therapy can induce immune tolerance in HA mice, but infusing platelets containing FVIII neither triggers anti-FVIII immune responses nor induces immune tolerance.

Aims: To investigate the modulatory function of platelets containing FVIII in FVIII immune responses.

Methods: Platelets were isolated from 2bF8 transgenic (2bF8Tg) mice in which FVIII expression was driven by the platelet-specific αIIb promoter. 2bF8Tg platelets with or without desialylation or platelet lysates were infused into HA mice, followed by FVIII immunization, and inhibitor titers were determined by Bethesda assay.

Results: First, we investigated the effect of co-infusion of 2bF8Tg platelets with recombinant human FVIII (rhFVIII) on inhibitor development. The inhibitor titer was 8-fold lower in HA mice that were co-infused with 2bF8Tg-platelets and rhFVIII compared to those treated with rhFVIII only (32±36 vs. 253±125 BU/ml, respectively). Co-infusion of acidified 2bF8Tg-platelet lysates with rhFVIII also significantly attenuated inhibitor titers in HA mice. Since desialylation of GPIbα plays a pivotal role in aged platelet apoptosis and targeting gene expression in apoptotic cells can induce immune tolerance, we tackled if desialylated platelets (dPlts) could modulate FVIII immune responses in HA mice. Platelets isolated from 2bF8Tg transgenic mice were treated with sialidase, resulting in 98% of sialic acid being removed from the end of the glycan on GPIbα. Infusion of 2bF8Tg-dPlts significantly increased Treg cells in HA mice, without inhibitor development. When dPlt-sensitized animals were immunized with rhFVIII, the incidence of inhibitor development and the titer significantly decreased compared to those in unsensitized animals under the same challenge.

Conclusion(s): FVIII-engineered platelets could be used to modulate FVIII immune responses. Infusion of 2bF8Tg-dPlts or lysates induces immune tolerance in HA mice.