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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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A panel of 147–155TX minigene-expressing vac recombinants containing random amino acid replacements at the COOH-terminal X position are all transported, processed by ACE, and presented by intact target cells. L-Kd cells were infected for 4 h with 5 PFU of the  indicated vac with 5 PFU of control virus (open bars), or with 5 PFU ACE  vac (solid bars), or cytosolically delivered ACE vac (hatched bars). 51Cr-labeled  target cells were then assessed for recognition by NP147–155-specific CTL  at an effector/target ratio of 50:1. Control virus in these experiments was  NP296–498 vac.
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Figure 2: A panel of 147–155TX minigene-expressing vac recombinants containing random amino acid replacements at the COOH-terminal X position are all transported, processed by ACE, and presented by intact target cells. L-Kd cells were infected for 4 h with 5 PFU of the indicated vac with 5 PFU of control virus (open bars), or with 5 PFU ACE vac (solid bars), or cytosolically delivered ACE vac (hatched bars). 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTL at an effector/target ratio of 50:1. Control virus in these experiments was NP296–498 vac.

Mentions: None of these constructs was presented in the absence of ACE, reiterating severe limitations on available mono- or dipeptidyl carboxypeptidase activity suggested by the TG block of presentation (Fig. 2). When coexpressed with ACE, every TX vac sensitized target cells for lysis by NP-specific CTLs. One trivial explanation for this phenomenon is that the COOH-terminal (X) residue may be cleaved in the cytosol, followed by transport and unconventional ACE-mediated removal of the remaining threonine. To address this possibility, we generated and tested a vac recombinant that expressed 147–155T (with no X residue). This construct failed to sensitize target cells for NP-specific lysis above background levels, and its presentation could not be rescued by ACE. Also shown in this figure, a form of ACE intentionally delivered to the cytosol failed to process TG and all other TX constructs tested. Taken with the previous finding that cytosolically delivered ACE is rapidly degraded and all detectable intracellular wild-type ACE activity segregates with the glycosylated form of the enzyme (27), this result indicates that the effects seen in our system are not due to anomalous expression of small amounts of ACE within the cytosol, and is consistent with a strict requirement for transport to the exocytic compartment. Thus, each member of the panel is transported sufficiently well within an intact cell to stimulate recognition and lysis by class I–restricted CTLs in an optimized chromium release assay.


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)

A panel of 147–155TX minigene-expressing vac recombinants containing random amino acid replacements at the COOH-terminal X position are all transported, processed by ACE, and presented by intact target cells. L-Kd cells were infected for 4 h with 5 PFU of the  indicated vac with 5 PFU of control virus (open bars), or with 5 PFU ACE  vac (solid bars), or cytosolically delivered ACE vac (hatched bars). 51Cr-labeled  target cells were then assessed for recognition by NP147–155-specific CTL  at an effector/target ratio of 50:1. Control virus in these experiments was  NP296–498 vac.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: A panel of 147–155TX minigene-expressing vac recombinants containing random amino acid replacements at the COOH-terminal X position are all transported, processed by ACE, and presented by intact target cells. L-Kd cells were infected for 4 h with 5 PFU of the indicated vac with 5 PFU of control virus (open bars), or with 5 PFU ACE vac (solid bars), or cytosolically delivered ACE vac (hatched bars). 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTL at an effector/target ratio of 50:1. Control virus in these experiments was NP296–498 vac.
Mentions: None of these constructs was presented in the absence of ACE, reiterating severe limitations on available mono- or dipeptidyl carboxypeptidase activity suggested by the TG block of presentation (Fig. 2). When coexpressed with ACE, every TX vac sensitized target cells for lysis by NP-specific CTLs. One trivial explanation for this phenomenon is that the COOH-terminal (X) residue may be cleaved in the cytosol, followed by transport and unconventional ACE-mediated removal of the remaining threonine. To address this possibility, we generated and tested a vac recombinant that expressed 147–155T (with no X residue). This construct failed to sensitize target cells for NP-specific lysis above background levels, and its presentation could not be rescued by ACE. Also shown in this figure, a form of ACE intentionally delivered to the cytosol failed to process TG and all other TX constructs tested. Taken with the previous finding that cytosolically delivered ACE is rapidly degraded and all detectable intracellular wild-type ACE activity segregates with the glycosylated form of the enzyme (27), this result indicates that the effects seen in our system are not due to anomalous expression of small amounts of ACE within the cytosol, and is consistent with a strict requirement for transport to the exocytic compartment. Thus, each member of the panel is transported sufficiently well within an intact cell to stimulate recognition and lysis by class I–restricted CTLs in an optimized chromium release assay.

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