Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation

Céline Vandamme,Johanne Le Duff,Marie Devaux,Mickaël Guilbaud,Célia Couzinié,Philippe Moullier,Xavier Saulquin,Oumeya Adjali,Leslie Hesnard,Rebecca Xicluna,Nicolas Jaulin

doi:10.3389/fimmu.2019.03110

Abstract

Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8+ T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8+ T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8+ T cells with a CD45RA+ CCR7− terminally-differentiated effector memory cell (TEMRA) fraction. Ex vivo frequencies of total AAV-specific CD8+ T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of TEMRA cells and IFNγ ELISpot positive responses. TEMRA cells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.

Highlights

Over the past decade, recombinant adeno-associated virus-derived vectors have emerged as a powerful vector platform for in vivo gene delivery
Following Tetramer-Associated Magnetic Enrichment (TAME), we were able to readily detect CD8+ T cells stained with pAAV2/A2 tetramers ex vivo in all A2+ donors tested (n = 15, Figure 2A)
Phenotypical characterization of Associated Viruses (AAV)-specific CD8+ T cells has almost exclusively relied on expansion or stimulation of peripheral blood mononuclear cells (PBMCs) or splenocytes prior to phenotypical or functional assessment [10, 17, 18]

Summary

Introduction

Recombinant adeno-associated virus-derived vectors (rAAV) have emerged as a powerful vector platform for in vivo gene delivery. Already three different AAV-based gene therapy products have received market approval [Glybera [6], Luxturna [7], Zolgensma [8]]. All these successes have been tempered by rising concerns over the immunogenicity of the AAV capsid in patients, especially when the vector was delivered via a systemic route. While the prevalence of anti-AAV antibodies among the human population is widely studied today [11], and their impact on rAAV-mediated gene transfer is fairly welldocumented [12], the detection and characterization of AAVspecific T cell responses remain somewhat more of a challenge even if this issue was first addressed more than 15 years ago [13]

Methods

Results

Conclusion