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J. Exp. Med.,
Volume 186, Number 10, November 17, 1997 1655-1662
By
From the * Kimmel Cancer Institute and 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.
Department of Biochemistry and Molecular Pharmacology,
Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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