title: Cross-presentation of human melanoma peptide antigen MART-1 to CTLs from in vitro reconstituted gp96/MART-1 complexes.
abstract: Heat shock proteins (HSPs) have two unique roles as constituents of tumor vaccines: (i) to shuttle associated tumor antigens into professional antigen-presenting cells (APCs) and (ii) to activate professional APCs. Here we investigated the shuttle function of the HSP gp96 (glycoprotein 96) for a human melanoma peptide antigen MART-1 that was noncovalently bound to gp96 in vitro. This in vitro complexing reaction was optimized using the radioiodinated MART-1 peptide and human gp96. Up to 20% of gp96 molecules could bind the peptide, assuming a 1:1 molar ratio. The binding was temperature-dependent and thus reversible. At -20 degrees C, 95% of the peptide remained complexed after 24 h, but 25% and 60% of the peptide dissociated at 37 degrees C within 6 and 24 h, respectively. This observation suggests that under the physiological conditions in APCs, spontaneous peptide dissociation from gp96 complexes may facilitate the delivery of peptide antigen into antigen presentation pathways. The gp96/MART-1 complexes stimulated an HLA A2-restricted MART-1-specific CTL clone dependent on the amount of complexed peptide and the presence of HLA-A2-positive APCs. The reaction was peptide-specific and could be blocked by an excess of untreated native gp96. These results show for the first time that peptide antigens from in vitro reconstituted gp96/peptide antigen complexes can be cross-presented by human APCs. These findings extend the scientific basis for further evaluating the use of either endogenous or in vitro reconstituted gp96/tumor-antigen complexes as tumor vaccines.
authors.affiliation: First Department of Internal Medicine, Johannes Gutenberg-University of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Staib
authors.forename: Martin
authors.initials: M
authors.lastname: Distler
authors.forename: Karen
authors.initials: K
authors.lastname: Bethke
authors.forename: Ute
authors.initials: U
authors.lastname: Schmitt
authors.forename: Peter R
authors.initials: PR
authors.lastname: Galle
authors.forename: Michael
authors.initials: M
authors.lastname: Heike
mesh_terms: D017951:Antigen Presentation; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D002461:Cell Line, Transformed; D002999:Clone Cells; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation; D058965:MART-1 Antigen; D046911:Macromolecular Substances; D009363:Neoplasm Proteins / Q000276:immunology* / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013696:Temperature
chemical_list: D000951:Antigens, Neoplasm; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D058965:MART-1 Antigen; C105787:MART-1-Melan-A(27-35) epitope; C548667:MLANA protein, human; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; C052646:sarcoma glycoprotein gp96 rejection antigens