Quantifying Capsid Peptide:MHC I Complexes Following Adeno-Associated Virus (AAV) Transduction

Recent data from a clinical trial of adeno-associated virus (AAV) 2-mediated hepatic gene transfer of factor IX into hemophilia B subjects demonstrated the presence of AAV2-capsid-specific CD8⁺ T cells and identified an immunodominant AAV2 epitope. The expansion and contraction of this CD8⁺ T cell population coincided with the rise and fall of an asymptomatic elevation in liver transaminases, and correlated with the expression and subsequent loss of factor IX expression. These observations suggest that CD8⁺ T cells directed against AAV capsid antigen clear the transduced hepatocytes. Indeed, others in our laboratory have directly shown the lysis of AAV-transduced hepatocytes by CD8⁺ T cells in vitro. However, the stoichiometry and kinetics of AAV capsid degradation and processing for MHC class I presentation are presently unknown. We now report the generation of soluble AAV capsid-specific T cell receptor (TCR) tetramers and their use in quantifying capsid peptide:MHC I complexes presented on cell surfaces. CD8⁺ T cell clones specific for AAV2-derived peptide 74 (VPQYGYLTL) presented in the context of HLA-B*0702 were isolated and used to generate TCR cDNA. This cDNA was transfected into CHO cells to produce soluble TCR α and β chain fusion proteins. The soluble TCR was biotinylated, then tetramerized to streptavidin-conjugated fluorochrome for use in flow cytometric analyses. Cell lines expressing HLA-B*0702 were loaded with cognate AAV2 peptide, a cross-reactive AAV1 peptide (IPQYGYLTL), or irrelevant HLA-B*0702 epitopes derived from HIV-1 gp120 (IPRRIRGGL) or EBV nuclear antigen (RPPIFIRRL). Staining with TCR-tetramers reveals that this reagent binds AAV-derived peptides specifically. In addition, this reagent exhibits HLA-specificity, as cognate peptide loaded onto cells which express alternate HLA alleles was not detected. These results demonstrate that TCR-tetramers retain their antigen and MHC context specificities. Capsid peptide titration experiments using HLA-B*0702-bearing JYA2B7 and SK-MES-1 cell lines reveal that TCR-tetramers exhibit a limit of detection of approximately 10⁵ cell surface peptide:MHC I complexes. Efficiency of detection is estimated at 1 peptide:MHC I complex for every 10¹⁰ peptide molecules added to cell culture. Preliminary studies indicate that the number of presenting complexes wanes by 48 hours following peptide loading. In contrast, AAV1-mediated transduction experiments demonstrate that capsid peptide:MHC I complexes are detectable after 48 hrs. In conclusion, these results are in agreement with our central hypothesis that AAV capsid is degraded upon transduction and gains access to MHC Class I antigen presentation pathways. Studies with the TCR-tetramers will allow definition of kinetics and capsid dose-dependence of antigen presentation, defining parameters of cell vulnerability to host immune-mediated clearance of transduced cells.