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.

中文翻译：

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基于四聚体的人类供体中现有抗AAV8 CD8 + T细胞的富集允许检测TEMRA亚群。

预先存在的对AAV衣壳的免疫力可能会危及rAAV介导的基因转移患者的安全性和效率。由于缺乏循环AAV特异性CD8 + T淋巴细胞的稀缺性，抗衣壳细胞毒性免疫反应已被证明是表征的挑战，而传统的流式细胞仪或ELISpot分析很少能检测到这种抗体。在这里，我们使用荧光MHC I类四聚体与磁富集相结合来检测人PBMC中的AAV8特异性CD8 + T细胞并表型，而无需事先扩增。我们显示，所有接受测试的健康个体均携带AAV8特异性CD8 + T细胞库，并带有CD45RA + CCR7-末端分化的效应记忆细胞（TEMRA）级分。总的AAV特异性CD8 + T细胞的离体频率不能预测IFNγELISpot反应，但有趣的是，我们证明了TEMRA细胞比例与IFNγELISpot阳性反应之间存在相关性。然后，TEMRA细胞可能在具有先前免疫功能的患者中在重组AAV介导的细胞毒性中发挥作用。总体而言，我们的结果鼓励开发新方法，结合rAAV介导的临床试验的观点，结合提高AAV特异性T细胞的检测灵敏度及其多功能评估，以更好地表征和监测AAV衣壳特异性细胞免疫反应。