MURINE TRANSPORTER ASSOCIATED WITH ANTIGEN PRESENTATION (TAP) PREFERENCES INFLUENCE CLASS I-RESTRICTED T-CELL RESPONSES

The transporter associated with antigen presentation (TAP) complex shu ttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Bioc hemical studies of murine and human TAP have established that substrat e length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities i n the intact cell and their influences on T cell responses have not be en demonstrated. We have devised a method for studying TAP-mediated tr ansport in intact cells, using T cell activation as a readout. The app roach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the K-d-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended wi th a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhib it vastly different transport capabilities in streptolysin O-permeabil ized cells, in accordance with thr predicted influence of the COOH-ter minal 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 converti ng 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, whe n T cell recognition was limited, either by blocking CD8 coreceptor in teractions or by decreasing the amount of transport substrate synthesi zed, significant COOH-terminal effects were revealed. Under such condi tions, those peptides that transported poorly in biochemical assays we re less efficiently presented. Therefore, TAP specificity operates in tile intact cell, appears to reflect previously defined rules with reg ard to the influence of thr COOH-terminal residue, and can strongly in fluence T cell responses.