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 cellpenetrating HIV TAT domain) bound in complexes with a tolllike 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.Abbreviations: CPP, cell-penetrating peptide; CPZ, chlorpromazine DC, dendritic cell; EEA, early endosomal antigen; EIPA, 5-(N-ethyl-Nisopropyl)amiloride; HLA, human leucocyte antigen; HUVEC, human umbilical vein endothelial cell; MbCD, methyl-b-cyclodextrin; MHC, major histocompatibility complex; OVA, ovalbumin; PBMC, peripheral blood mononuclear cell; Poly(i/c), polyinosinic/polycytidylic acid Th1, T helper cell type 1.

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells

“…It is now becoming apparent that the uptake could also be via endocytosis [12,37]. To ascertain the mechanism of OVA presentation we have used various inhibitors that can prevent uptake and thereby interfering with the processing and presentation of AntpOVA by DC.…”

“…Membrane translocating peptides such as the peptide derived from the antennapedia homeodomain or HIV TAT protein have been used to shuttle proteins or peptides into the cytoplasm of antigen presenting cells [13,14,16,[18][19][20]23,24,26,28,30,37,65].…”

“…This interaction is solely due to an ionic interaction between the positively charged CPP and the negatively charged receptor. The mechanism of antigen presentation of TATand penetratin-based peptide immunogens have been investigated however, there have been no mechanistic studies with penetratin-based whole proteins such as OVA [16,20,30,37].…”

Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity

“…To compare the ability of CPPs to enhance the immunogenicity of peptide vaccines, we selected eight previously reported sequences that ranged in their hydrophobicity, net charge, length, and origin. We included several CPPs that have been previously reported to augment peptide vaccines: penetratin (pAntp) ( 36 , 39 ), HIV transactivator of transcription (Tat) ( 38 , 44 ), Arg 8 ( 45 ), and MPG ( 46 ). Additionally, this series included CPPs that, to our knowledge, have never been tested for vaccine antigen delivery: Bpep ( 29 ), Diatos peptide vector 6 (DPV6) ( 47 ), vascular endothelial cadherin-derived peptide (pVEC) ( 48 ), and Transportan 10 (TP10) ( 49 ).…”

NickFect type of cell-penetrating peptides present enhanced efficiency for microRNA-146a delivery into dendritic cells and during skin inflammation

“…Although immunogenic responses might be of interest for enhancing immune system responses in immunotherapies for cancer or infectious diseases; they are totally undesirable in case of therapies aimed for attenuating inflammatory and autoimmune responses. It has been reported that some peptides with significant aggregation could affect immunogenicity as it is known that larger particles are more efficiently phagocytised and presented at the membrane level 53 . Activated DCs undergo morphological changes by developing extensions that increase cellular surface area to improve interaction with T cells, as well as upregulate major histocompatibility complex and costimulatory molecules such as CD86 54 .…”