Exposure to factor VIII protein in the presence of phosphatidylserine induces hypo-responsiveness toward factor VIII challenge in hemophilia A mice

Abstract

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4(+) regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-β antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.

Keywords: Biotechnology; Blood Coagulation Factors; Dendritic Cells; Factor VIII; GFP-FoxP3; Immunological Tolerance; Immunosuppression; Phosphatidylserine; Regulatory T-cell; Reverse/Inverse Vaccination.

FIGURE 1.

Phosphatidylserine complex induces hypo-responsiveness, but animals respond normally to irrelevant antigen. A, anti-FVIII Nab titer levels on the 11th week. The dotted horizontal line indicates the lowest observed free FVIII titer level. BU, Bethesda units. Std. Dev., standard deviation. B and C, development of anti-FVIII Nab (B) and anti-Ova immune response (C) upon administration of FVIII or FVIII-PS/PC along with ovalbumin. The large asterisks and the stars represent individual data points for the groups FVIII-PG and FVIII + Dex, respectively. kU, kilounits.

FIGURE 2.

The PS-mediated hypo-responsiveness involves Tregs. A, adoptive Transfer Study: anti-FVIII Nab titers (mean ± S.E.) following adoptive transfer of splenic CD4⁺CD25⁺ T-cells from FVIII or FVIII-PS/PG or naive (previously untreated) HA mice into naive, recipient HA mice and rechallenged with free FVIII. BU, Bethesda units. B, bar (left panel) and dot plots (middle panel, FVIII; right panel, FVIII-PS) of the percentage of FoxP3⁺ cells isolated from splenocytes of hemophilia A mice immunized with FVIII and FVIII-PS. C, dot plots of FoxP3 cells as measured by GFP expression in isolated lymph nodes of Treg hemophilia A mice (GFP knock-in FoxP3 FVIII^−/− mice) in naive mice (panel a) and in mice immunized with FVIII (panel b) and FVIII-PS (in the absence (panel c) and in the presence (panel d) of anti-TGF-β). SSC-H denotes the side scatter. D, total anti-FVIII titers measured in Treg hemophilia A mice following immunization with FVIII, FVIII-PS, and FVIII-PS+anti-TGF-β.