Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells

Background: We previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIII^null mice transplanted with 2bF8-transduced Sca-1⁺ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost. Objective: In this study, we aimed to describe the mechanism of this platelet-FVIII loss. Methods: We monitored platelet-FVIII expression in FVIII^null mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1⁺ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8^Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8^Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell–deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity. Results: Platelet-FVIII expression was sustained in 2bF8^Tg bone marrow transplantation but not in 2bF8^Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8^Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8^Tg-Sca-1⁺ cells and secreted interferon gamma, but were not stimulated by 2bF8^Tg platelets/megakaryocytes, suggesting that 2bF8^Tg-Sca-1⁺ cells are targets for FVIII-primed CD8 T cells. When 2bF8^Tg-Sca-1⁺ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice. Conclusion: Pre-existng immunity can alter the engraftment of 2bF8^Tg-Sca-1⁺ cells through the cytotoxic CD8 T-cell–mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.