Buhl Timo, Braun Andrea, Forkel Susann, Möbius Wiebke, van Werven Lars, Jahn Olaf, Rezaei-Ghaleh Nasrollah, Zweckstetter Markus, Mempel Martin, Schön Michael P, Haenssle Holger A
Clinic of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany.
Exp Dermatol. 2014 Jan;23(1):20-6. doi: 10.1111/exd.12285.
Optimized delivery of antigens combined with sustainable maturation of dendritic cells (DCs) is crucial for generation of effective antitumoral immune responses. Multiple approaches for ex vivo antigen loading and improvement in immunogenicity have been described. We have recently established a single-step protocol consisting of a fusion peptide (a sequence of the melanoma antigen Melan-A and a cationic cell-penetrating HIV TAT domain) bound in complexes with a toll-like receptor agonist. As the exact cellular uptake mechanisms of TAT-coupled antigens have been a matter of considerable debate and significantly depend on cell type, cargo and concentrations, we evaluated internalization routes into human immature DCs in comparison with non-phagocytic cell lines. We found that Melan-A-TAT fusion peptide uptake by DCs is mainly energy dependent, superior compared with polylysine-coupled Melan-A and significantly higher in DCs as compared with Jurkat cells or HUVECs. Furthermore, we could track the uptake of the fusion peptide exclusively through early endosomes to lysosome compartments after 90 min by fluorescence microscopy and immunoelectron microscopy. Specific endocytosis inhibitors revealed major internalization of the fusion peptide by DCs via clathrin-mediated endocytosis, whereas uptake by non-phagocytic HUVECs differed significantly, involving macropinocytosis as well as clathrin-mediated endocytosis. As our understanding of the processes involved in internalization of TAT-coupled cargos by human DCs broadens, and DC activation becomes available by single-step procedures as described, further development of simultaneous DC maturation and intra-cellular peptide targeting is warranted.
优化抗原递送并结合树突状细胞（DCs）的可持续成熟对于产生有效的抗肿瘤免疫反应至关重要。已经描述了多种用于体外抗原加载和提高免疫原性的方法。我们最近建立了一个单步方案，该方案由一种融合肽（黑色素瘤抗原Melan-A的序列和一个阳离子细胞穿透性HIV TAT结构域）与一种Toll样受体激动剂结合形成复合物组成。由于TAT偶联抗原的确切细胞摄取机制一直存在相当大的争议，并且显著取决于细胞类型、货物和浓度，我们将其与非吞噬细胞系进行比较，评估了其进入人未成熟DCs的内化途径。我们发现，DCs对Melan-A-TAT融合肽的摄取主要依赖能量，与多聚赖氨酸偶联的Melan-A相比更具优势，并且与Jurkat细胞或人脐静脉内皮细胞（HUVECs）相比，在DCs中的摄取显著更高。此外，通过荧光显微镜和免疫电子显微镜，我们可以在90分钟后追踪融合肽仅通过早期内体进入溶酶体区室的摄取过程。特异性内吞抑制剂显示，DCs通过网格蛋白介导的内吞作用主要内化融合肽，而非吞噬性HUVECs的摄取则有显著差异，涉及巨胞饮作用以及网格蛋白介导的内吞作用。随着我们对人DCs摄取TAT偶联货物所涉及过程的理解不断拓宽，并且如所述通过单步程序可以实现DC激活，同时进行DC成熟和细胞内肽靶向的进一步发展是有必要的。
