Sarukhan A, Soudais C, Danos O, Jooss K
Institut Necker, INSERM 345, Paris, France.
J Gene Med. 2001 May-Jun;3(3):260-70. doi: 10.1002/jgm.175.
We have previously demonstrated that recombinant adeno-associated virus vectors expressing the influenza virus hemagglutinin (rAAV-HA) in skeletal muscle results in T-cell priming and muscle fiber destruction due to cross-presentation of HA by dendritic cells (DC). Based on controversial observations concerning the stability of non-self proteins expressed from rAAV vectors it is important to understand the factors influencing cross-presentation of transgene products following rAAV mediated gene transfer, in order to be able to use this vector safely in the clinic.
In order to understand the factors influencing in vivo cross-presentation of non-self proteins, we have retargeted the immunogenic lacZ protein in the context of rAAV from the cytoplasm to the cell surface and studied the activation of LacZ specific immune responses following intramuscular mediated gene transfer. In addition, using tools available for studying in vitro HA-specific T-cell activation, our aim was to identify the cell types involved in class I and class II restricted cross-presentation as well as the nature of the cross-presented material.
By retargeting the lacZ protein in the context of rAAV to the cell membrane, we found that one of the factors influencing the efficiency of cross-presentation of non-self antigens is the localization of the transgene product within the target cells. Following rAAV-LacZ mediated gene transfer to the muscle we demonstrated that the membrane-bound form of LacZ resulted in target cell destruction, which is in stark contrast to the stability observed with rAAV-LacZ vectors expressing the cytoplasmic form of LacZ. Using an in vitro assay, we were able to show that dendritic cells (DC) in addition to B-cells cross-presented HA to class II restricted T-cells whereas only the former were able to activate class I restricted CD8+ T-cells. High-dose antigens were needed for efficient class I restricted T-cell priming, whereas class II restricted T-cells were activated by less antigen.
The present results indicate that immune responses to non-self antigens expressed from rAAV vectors depend on the accessibility of such antigens to different local antigen-presenting cells.
我们之前已经证明，在骨骼肌中表达流感病毒血凝素的重组腺相关病毒载体（rAAV-HA）会导致T细胞致敏以及由于树突状细胞（DC）对HA的交叉呈递而造成肌纤维破坏。基于关于rAAV载体表达的非自身蛋白稳定性的有争议的观察结果，了解影响rAAV介导的基因转移后转基因产物交叉呈递的因素很重要，以便能够在临床上安全地使用这种载体。
为了了解影响非自身蛋白体内交叉呈递的因素，我们将rAAV背景下具有免疫原性的lacZ蛋白从细胞质重新定位到细胞表面，并研究了肌肉内介导的基因转移后LacZ特异性免疫反应的激活情况。此外，利用可用于研究体外HA特异性T细胞激活的工具，我们的目的是确定参与I类和II类限制性交叉呈递的细胞类型以及交叉呈递物质的性质。
通过将rAAV背景下的lacZ蛋白重新定位到细胞膜，我们发现影响非自身抗原交叉呈递效率的因素之一是转基因产物在靶细胞内的定位。在rAAV-LacZ介导的基因转移到肌肉后，我们证明LacZ的膜结合形式导致靶细胞破坏，这与表达细胞质形式LacZ的rAAV-LacZ载体所观察到的稳定性形成鲜明对比。使用体外试验，我们能够表明除了B细胞外，树突状细胞（DC）将HA交叉呈递给II类限制性T细胞，而只有前者能够激活I类限制性CD8+ T细胞。高效的I类限制性T细胞致敏需要高剂量抗原，而II类限制性T细胞则由较少的抗原激活。
目前的结果表明，对rAAV载体表达的非自身抗原的免疫反应取决于此类抗原对不同局部抗原呈递细胞的可及性。
