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Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

Yellen-Shaw AJ, Laughlin CE, Metrione RM, Eisenlohr LC - J. Exp. Med. (1997)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

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.

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Schematic diagram  of transport-dependent ACE-mediated processing in the exocytic compartment.
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Figure 1: Schematic diagram of transport-dependent ACE-mediated processing in the exocytic compartment.

Mentions: In this study, we describe a unique approach to measuring the influence of TAP specificity within live, intact cells, using the physiologically significant readout of CTL recognition. A previous study demonstrated that the Kd-restricted influenza nucleoprotein epitope, amino acids 147–155, cannot be processed and presented when appended by two COOH-terminal residues, threonine and glycine, due to the unavailability of appropriate proteolytic activity (21). Presentation of the epitope can take place when the fragment is coexpressed with the dipeptidyl carboxypeptidase angiotensin converting enzyme (ACE; references 26 and 27). As rescue is intracellular and TAP-dependent, and this enzyme is active only in the exocytic compartment, and ACE-mediated processing of the 11-mer is a reflection of TAP-mediated transport within intact target cells (see Fig. 1 for a schematic of this process). We have used this system to examine class I–restricted presentation of an array of substrates that differ significantly in their in vitro transport capabilities.


Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

Yellen-Shaw AJ, Laughlin CE, Metrione RM, Eisenlohr LC - J. Exp. Med. (1997)

Schematic diagram  of transport-dependent ACE-mediated processing in the exocytic compartment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199128&req=5

Figure 1: Schematic diagram of transport-dependent ACE-mediated processing in the exocytic compartment.
Mentions: In this study, we describe a unique approach to measuring the influence of TAP specificity within live, intact cells, using the physiologically significant readout of CTL recognition. A previous study demonstrated that the Kd-restricted influenza nucleoprotein epitope, amino acids 147–155, cannot be processed and presented when appended by two COOH-terminal residues, threonine and glycine, due to the unavailability of appropriate proteolytic activity (21). Presentation of the epitope can take place when the fragment is coexpressed with the dipeptidyl carboxypeptidase angiotensin converting enzyme (ACE; references 26 and 27). As rescue is intracellular and TAP-dependent, and this enzyme is active only in the exocytic compartment, and ACE-mediated processing of the 11-mer is a reflection of TAP-mediated transport within intact target cells (see Fig. 1 for a schematic of this process). We have used this system to examine class I–restricted presentation of an array of substrates that differ significantly in their in vitro transport capabilities.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

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.

Show MeSH
Related in: MedlinePlus