697. Phenotypic and Functional Characterisation of Human Anti-AAV CD8+ T Cells Using MHC Class I Tetramer-Associated Magnetic Enrichment
Authors: Celine Vandamme, Marie Devaux, Nicolas Jaulin, Mickaël Guilbaud, Johanne Le Duff, Leslie Hesnard, Hanane Djamai, Marc Bonneville, Philippe Moullier, Xavier Saulquin, Oumeya Adjali
Journal: Molecular Therapy (2016)

Introduction: Recombinant Adeno-Associated Vectors (AAV) represent nowadays the most largely used platform for in vivo gene therapy. Unfortunately, despite promising results in preclinical and clinical studies, pre-existing immunity against the viral capsid remains one of the major hurdles to the safety and efficiency of AAV-based gene transfer in Humans. Particularly, the impact of pre-existing anti-AAV CD8+ T lymphocytes on gene transfer remains poorly defined. This gap in knowledge can, in part, be attributed to the scarcity of AAV-specific CD8+ T cells among the peripheral blood mononuclear cells (PBMCs), and to the lack of relevant animal models. Experimental approach: In order to set up a more sensitive and comprehensive method to detect and characterise AAV-specific CD8+ T cells among PBMCs, we used Tetramer-Associated Magnetic Enrichment (TAME) to analyse the frequency and phenotype of AAV-specific CD8+ T cells by flow cytometry. To this end, we previously generated AAV-loaded tetramers using the UV-mediated peptide exchange technology. Results: We were able to detect AAV8 and AAV2 capsid-specific CD8+ T cells among PBMCs in all healthy donors tested (n>40), without any amplification, at frequencies ranging from 1.10−6 to 1.10−4 CD8+ T cells. Phenotypic assessment of the detected cells revealed a small proportion of memory CD45RO+ cells, a population that expectedly could have emerged after primary infection with wild-type AAV. Though AAV-specific CD8+ T cells were detectable by TAME in all donors tested, only few of them (11/42) had a positive response when cellular responses in PBMCs were assessed by anti-AAV IFN-γ ELISpot assays. Interestingly, we registered higher ex vivo frequencies and more frequent positive ELISpot responses in HLA-B7 donors then in HLA-A2's. Evaluation of anti-AAV antibody and neutralizing factor titres in the sera of the same donors revealed no correlation between humoral and cellular responses, as has been previously described. To further the functional assessment of the detected cells, we sorted AAV8 capsid-specific CD8+ T cells after TAME and expanded human primary T cell lines. Purity of the cell lines was checked using tetramer staining. We succeeded in generating several functional AAV-specific CD8+ T cell lines that upregulated CD107 and secreted IFN-γ, TNF-α, Granzyme B and Perforin when faced with AAV8-loaded target cells. Interestingly, some cell lines seemed to display a lack of reactivity that could not be attributed to a generally compromised functionality, suggesting that a fine tuning of AAV-specific CD8+ T cells' activation might come into play, and is still under investigation. Conclusions and Perspectives: The dissimilarities observed ex vivo between humoral and cellular responses, as well as the different activation patterns registered in vitro highlight the need to gather multiple insights on capsid immunogenicity to better understand the onset of pre-existing anti-AAV immunity on recombinant AAV-based gene transfer and its impact on clinical outcome.