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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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ACE-mediated rescue under conditions of limited starting  substrate. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated  vacs alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed  for recognition by NP147–155-specific CTLs. Shown are two independent  experiments. Effector/target ratios were 30:1 for experiment 1 and 28:1  for experiment 2.
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Figure 7: ACE-mediated rescue under conditions of limited starting substrate. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated vacs alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTLs. Shown are two independent experiments. Effector/target ratios were 30:1 for experiment 1 and 28:1 for experiment 2.

Mentions: The panel of TX constructs was placed behind each of these structures, recombined into vaccinia, and tested for ACE-mediated presentation. We failed to see significant presentation of any of the constructs when placed behind HP20 and thus concentrated our analysis on the three less stringent barrier hairpins. As shown in Fig. 7, presentation efficiencies of the constructs across the range of barriers were consistent with the biochemical and anti-CD8 analyses. For example, the well-transported constructs, TM and TL, sensitized targets suboptimally when placed behind both the HP18 and the HP16 barriers. Both achieved optimal presentation when placed behind HP14, the least thermostable of the hairpin structures. TP, a poorly transported substrate in vitro, exhibited significantly lower presentation levels in all of the hairpin contexts. In experiment 2, presentation was still suboptimal in the HP14 context. Presentation of the substrate exhibiting no detectable transport in vitro, TG, was significantly lower in all contexts; presentation was still significantly suboptimal when placed behind HP14. In fact, in the experiments shown, even wild-type TG presentation was somewhat lower than the other constructs, although this was not consistently observed (see Fig. 2).


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)

ACE-mediated rescue under conditions of limited starting  substrate. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated  vacs alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed  for recognition by NP147–155-specific CTLs. Shown are two independent  experiments. Effector/target ratios were 30:1 for experiment 1 and 28:1  for experiment 2.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: ACE-mediated rescue under conditions of limited starting substrate. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated vacs alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTLs. Shown are two independent experiments. Effector/target ratios were 30:1 for experiment 1 and 28:1 for experiment 2.
Mentions: The panel of TX constructs was placed behind each of these structures, recombined into vaccinia, and tested for ACE-mediated presentation. We failed to see significant presentation of any of the constructs when placed behind HP20 and thus concentrated our analysis on the three less stringent barrier hairpins. As shown in Fig. 7, presentation efficiencies of the constructs across the range of barriers were consistent with the biochemical and anti-CD8 analyses. For example, the well-transported constructs, TM and TL, sensitized targets suboptimally when placed behind both the HP18 and the HP16 barriers. Both achieved optimal presentation when placed behind HP14, the least thermostable of the hairpin structures. TP, a poorly transported substrate in vitro, exhibited significantly lower presentation levels in all of the hairpin contexts. In experiment 2, presentation was still suboptimal in the HP14 context. Presentation of the substrate exhibiting no detectable transport in vitro, TG, was significantly lower in all contexts; presentation was still significantly suboptimal when placed behind HP14. In fact, in the experiments shown, even wild-type TG presentation was somewhat lower than the other constructs, although this was not consistently observed (see Fig. 2).

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