title: Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.
abstract: The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O-permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.
authors.affiliation: Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Yellen-Shaw
authors.forename: C E
authors.initials: CE
authors.lastname: Laughlin
authors.forename: R M
authors.initials: RM
authors.lastname: Metrione
authors.forename: L C
authors.initials: LC
authors.lastname: Eisenlohr
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D017951:Antigen Presentation; D016827:CD8 Antigens / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000276:immunology*; D007739:L Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D007703:Peptidyl-Dipeptidase A / Q000502:physiology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D014616:Vaccinia virus / Q000276:immunology
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D019138:Antibodies, Blocking; D016827:CD8 Antigens; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; D007703:Peptidyl-Dipeptidase A