High-Level Expression of an Allo-MHC II Transgene Can Be Achieved in Mouse Hepatocytes, But Lack of Chaperones and Accessory Molecules May Prevent Tolerance Induction.

Abstract

Background: Previous studies have shown induction of specific tolerance and long term survival of fully allogeneic cardiac grafts when donor MHC class II genes were transferred to recipient bone marrow prior to transplantation. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb-expressing skin grafts. Here, we investigate the effect of high level recombinant Adeno-Associated Virus (AAV)-mediated expression of MHC class II in heart transplant setting. Methods: We developed an efficient liver-specific vector encoding H-2IAd (rAAV-IAd). C57BL/6 (B6, H-2b) WT and Foxp3GFPmice were injected with varying doses (5x108-5x1011 vector genome copies, vgc) to produce expression of H-2IAd on hepatocytes. Expression of IAd and costimulatory molecules was assessed by FACS on isolated hepatocytes, whilst expression of Invariant chain (Ii) and H-2M was determined by RT-PCR. Fully-allogeneic hearts from DBA/2 (H-2d) were transplanted into C57BL/6 at d7 or d100 post-transduction. Results: High level expression of H-2IAd on hepatocytes of B6 mice with doses of 5x1010 or 5x1011 rAAV-IAdwas observed.Figure: No Caption available.Foxp3+ CD4+ T cells were increased in the livers of mice receiving 5x1011 rAAV-IAd with 2±0.76% in normal ctrl, 2.4± 0.6% for 5x1010 IAd injected mice and 7.7±2.2% for 5x1011 IAd injected mice (p<0.03).Yet, the survival rate of hearts grafted into rAAV-IAd treated B6 mice was not improved compared to grafts in normal mice (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1. In addition, RT-PCR revealed minimal expression of H2-M and Ii, suggesting an impaired function in peptide editing of expressed IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating an immune response and tolerance to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for biosynthesis of fully functional MHCII IAd from hepatocytes.