title: Internalization routes of cell-penetrating melanoma antigen peptides into human dendritic cells.
abstract: 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.
authors.affiliation: Clinic of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Buhl
authors.forename: Andrea
authors.initials: A
authors.lastname: Braun
authors.forename: Susann
authors.initials: S
authors.lastname: Forkel
authors.forename: Wiebke
authors.initials: W
authors.lastname: Möbius
authors.forename: Lars
authors.initials: L
authors.lastname: van Werven
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
authors.forename: Nasrollah
authors.initials: N
authors.lastname: Rezaei-Ghaleh
authors.forename: Markus
authors.initials: M
authors.lastname: Zweckstetter
authors.forename: Martin
authors.initials: M
authors.lastname: Mempel
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schön
authors.forename: Holger A
authors.initials: HA
authors.lastname: Haenssle
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002454:Cell Differentiation; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D019169:Jurkat Cells; D008247:Lysosomes / Q000378:metabolism; D058965:MART-1 Antigen / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D058965:MART-1 Antigen; C548667:MLANA protein, human; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000255:Adenosine Triphosphate
keywords: Melan-A/Mart-1; TAT; cell-penetrating peptides; cellular immunotherapy; dendritic cells