title: Intracellular delivery of major histocompatibility complex class I-binding epitopes: dendritic cells loaded and matured with cationic peptide/poly(I:C) complexes efficiently activate T cells.
abstract: Based on their role for the induction of T-cell responses, dendritic cells (DCs) are popular candidates in cancer vaccine development. We established a novel single-step intracellular delivery of peptide/poly(I:C) complexes for antigen loading and Toll-like receptor-3 (TLR3)-mediated maturation of human DCs using a cell-penetrating peptide (tat(49-57): RKKRRQRRR) as delivery vector. Towards this end, a cationic tat-sequence was fused with an antigenic, major histocompatibility complex (MHC) class I-binding melanoma epitope (Melan-A/Mart-1 sequence: ELAGIGILTV) and then mixed with negatively charged poly(I:C) dsRNA to form peptide/nucleic acid complexes. Flow cytometry and confocal laser scanning microscopy confirmed intracellular localization of TLR3 in monocyte-derived immature DCs (iDCs). Peptide/poly(I:C) complexes were readily internalized by iDCs without negatively affecting cell viability. They induced DC maturation and secretion of bioactive interleukin (IL)-12p70. When peptide/poly(I:C) complex-loaded DCs were used for autologous T cell stimulation, epitope-specific interferon-gamma secretion was quantitatively superior in comparison to peptide-loaded DCs matured by a cytokine cocktail, as detected by enzyme-linked immunospot assays. Thus, complexes of cationic antigenic peptides and poly(I:C) might be of great utility for a TLR3-mediated DC maturation and intracellular peptide targeting in a single step. Resulting DCs induce a strong expansion/activation of antigen-specific T cells in the context of an IL-12p70 secretion.
authors.affiliation: Department of Dermatology, Georg-August-University Göttingen, Göttingen, Germany. h.haenssle@med.uni-goettingen.de
authors.forename: Holger A
authors.initials: HA
authors.lastname: Haenssle
authors.forename: Petra
authors.initials: P
authors.lastname: Riedl
authors.forename: Timo
authors.initials: T
authors.lastname: Buhl
authors.forename: Anke
authors.initials: A
authors.lastname: Schardt
authors.forename: Albert
authors.initials: A
authors.lastname: Rosenberger
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schön
authors.forename: Reinhold
authors.initials: R
authors.lastname: Schirmbeck
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D002470:Cell Survival / Q000187:drug effects; D003713:Dendritic Cells / Q000187:drug effects* / Q000378:metabolism; D000939:Epitopes; D005805:Genes, MHC Class I; D006801:Humans; D007369:Interferon Inducers / Q000494:pharmacology*; D018664:Interleukin-12 / Q000378:metabolism; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology / Q000628:therapy; D009363:Neoplasm Proteins / Q000276:immunology*; D011070:Poly I-C / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000276:immunology*; D051196:Toll-Like Receptor 3 / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D000939:Epitopes; D007369:Interferon Inducers; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D054322:tat Gene Products, Human Immunodeficiency Virus; D018664:Interleukin-12; D011070:Poly I-C