Exosomes as a tumor vaccine: Enhancing potency through direct loading of antigenic peptides
Exosomes as a tumor vaccine: Enhancing potency through direct loading of antigenic peptides
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DOI：
10.1097/00002371-200309000-00007
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发表时间：
2003-09-01
影响因子：
3.9
通讯作者：
Le Pecq, JB
中科院分区：
文献类型：
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作者：
Hsu, DH;Paz, P;Le Pecq, JB
树突状细胞(DC)分泌的外体含有MHC-I、MHC-II和其他将抗原呈递给T细胞所需的辅助分子。以前的研究表明，通过向DC培养中添加多肽而间接负载的外切体MHC-I具有免疫原性。然而，没有进行多肽结合分析来将T细胞刺激活性与外体上MHC-I/多肽复合体的数量联系起来。在这项研究中，我们测量了不同负载条件下与MHC-I结合的多肽，并测试了外切体在T细胞激活实验中的效力。我们证明，纯化的外切体上的MHC-I可以直接负载比间接负载更高水平的多肽。在弱酸性条件下进行的直接加载方法即使在没有外源β2m的情况下也是有效的。这种多肽结合量的增加极大地增强了外切体的效力，使我们能够在体外进一步研究外切体的生物学活性。在抗原提呈细胞(APC)存在的情况下，直接负载人类白细胞抗原A2限制性MART1肿瘤多肽的外切体能刺激人类白细胞抗原A2/MART1特异性T细胞系。T细胞通过人类白细胞抗原A2(Neg)APC对外切体进行反应，证实了功能性MHC-I/多肽复合体的转移，而不仅仅是多肽转移到APC。在DC外切体上大量表达的MHC-II分子也在与MHC-I相同的条件下进行了功能负载。这一功能允许传递多种多肽抗原，既可以刺激CD8(+)细胞毒性T细胞，也可以刺激对有效抗肿瘤反应至关重要的CD4(+)T辅助细胞。优化的负载条件和将MHC-I和MHC-II抗原转移到APC的能力导致了外切体的发展，使其成为一种目前正在临床试验中的“无细胞”免疫治疗方法。
Exosomes secreted by dendritic cells (DCs) contain MHC-I, MHC-II, and other accessory molecules required for antigen presentation to T cells. Previous studies have shown that exosome MHC-I "indirectly" loaded by adding peptides to DC cultures are immunogenic. However, analysis of peptide binding was not performed to link T-cell-stimulating activity with the amount of MHC-I/peptide complexes on the exosomes. In this study, we measured peptide binding to MHC-I under different loading conditions and tested the exosomes' potencies in T-cell activation assays. We demonstrate that MHC-I on purified exosomes can be directly loaded with peptide at much greater levels than indirect loading. The direct loading method performed in mildly acidic conditions was effective even in the absence of exogenous beta2m. This increase in peptide binding greatly enhanced exosome potency, allowing us to further study the biologic activity of exosomes in vitro. In the presence of antigen-presenting cells (APC), exosomes directly loaded with the HLA-A2 restricted MART1 tumor peptide stimulated an HLA-A2/MART1 specific T-cell line. The T cells responded to exosomes using HLA-A2(neg) APC, demonstrating transfer of functional MHC-I/peptide complexes and not peptide alone to APC. MHC-II molecules, which are abundantly expressed on DC exosomes, were also functionally loaded under the same conditions as MHC-I. This feature allows for delivery of multiple peptide antigens that can stimulate both CD8(+) cytotoxic T cells as well CD4(+) T helper cells critical for an effective antitumor response. The optimized loading conditions and the ability to transfer both MHC-I and MHC-II antigens to APC have led to the development of exosomes as an "acelullar" immunotherapy approach currently being tested in clinical trials.