Overcoming Naïve and Memory Immune Responses in a Mouse Skin Transplant Model Using High-Level Expression of Donor MHC Antigen in Recipient Livers

Abstract

Introduction: The liver has long been recognised as having tolerogenic properties, especially in transplantation. Our previous work has demonstrated a number of factors involved, including the large size of the liver, its complement of immunosuppressive donor leucocytes and the ability of hepatocytes to directly interact with and destroy antigen-specific T cells. Methods: We developed a recombinant adeno-associated virus vector (rAAV-Kb) expressing H-2Kb under the control of liver-specific promoters and enhancers. Intravenous injection of (H-2Kk-expressing) B10.BR mice with 5 × 1010 viral genome copies (vgc) of rAAV-Kb rapidly produced wild-type levels of H-2Kb expression on all hepatocytes. Skin grafts from (H-2Kb-expressing) 178.3 donors were transplanted onto recipient B10. BR mice 7 days after rAAV-Kb treatment and graft survival was monitored. Results: Untreated B10.BR mice rejected their 178.3 grafts with a median survival time (MST) of 18.7 days (n=18). Grafts onto mice that had been treated with 5 × 1010 vgc rAAV-Kb had markedly prolonged survival (MST >180 days, n=17), comparable to syngeneic grafts (Figure 1).[Figure 1]Injection of primed animals (that had previously rejected a 178.3 skin graft) with 5 × 1010 vgc rAAV-Kb resulted in long term expression of H-2Kb and no liver pathology. Secondary grafts onto these primed recipients, subsequently treated with rAAV-Kb, had prolonged survival (MST >100 days, n=9) while grafts onto untreated primed animals showed accelerated rejection (MST 12 days), as expected (Figure 2).[Figure 2]In contrast to animals treated with the high-dose of rAAV-Kb (5 × 1010 vgc), recipients that received a low dose of rAAV-Kb (5 × 106 vgc) demonstrated an accelerated rate of rejection of 178.3 grafts (MST 11 days). Immunohistochemical analysis of skin grafts from mice treated with this low dose revealed increased expression of H-2Kb (MHC Class I) and ICAM-1 at days 7 and 14 post-transplant, compared to grafts from mice treated with the high dose. Conclusion: High-level expression of donor MHC antigen in recipient livers was sufficient to promote tolerance to skin grafts, even in primed animals. This demonstrates that hepatocyte-directed AAV vectors can be used as a novel approach for obtaining antigen-specific immunosuppression that can overcome a primed T cell memory response.