Premium
Internalization routes of cell‐penetrating melanoma antigen peptides into human dendritic cells
Author(s) -
Buhl Timo,
Braun Andrea,
Forkel Susann,
Möbius Wiebke,
Werven Lars,
Jahn Olaf,
RezaeiGhaleh Nasrollah,
Zweckstetter Markus,
Mempel Martin,
Schön Michael P.,
Haenssle Holger A.
Publication year - 2014
Publication title -
experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.108
H-Index - 96
eISSN - 1600-0625
pISSN - 0906-6705
DOI - 10.1111/exd.12285
Subject(s) - endocytosis , internalization , pinocytosis , microbiology and biotechnology , endosome , jurkat cells , antigen , cell penetrating peptide , biology , t cell , antigen presentation , chemistry , peptide , immune system , cell , immunology , biochemistry , intracellular
Optimized delivery of antigens combined with sustainable maturation of dendritic cells ( DC s) 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 M elan‐ 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 DC s in comparison with non‐phagocytic cell lines. We found that M elan‐ A ‐ TAT fusion peptide uptake by DC s is mainly energy dependent, superior compared with polylysine‐coupled M elan‐ A and significantly higher in DC s as compared with Jurkat cells or HUVEC s. 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 DC s via clathrin‐mediated endocytosis, whereas uptake by non‐phagocytic HUVEC s 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 DC s 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.