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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-rescued presentation under conditions of limited T cell  receptor stimulation. L-Kd cells were coinfected for 4 h with 5 PFU of  the indicated vac alone and 5 PFU ACE vac. 51Cr-labeled target cells  were then assessed for recognition by NP147–155-specific CTLs with or  without the indicated concentrations of anti-CD8 antibody 2.43. CTLs  were used at a constant effector/target ratio of 10:1.
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Figure 5: ACE-rescued presentation under conditions of limited T cell receptor stimulation. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated vac alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTLs with or without the indicated concentrations of anti-CD8 antibody 2.43. CTLs were used at a constant effector/target ratio of 10:1.

Mentions: When we examined presentation of the epitope from the various TX contexts following the addition of brefeldin A at numerous time points after infection, we failed to distinguish presentation differences between them (data not shown). However, the two other approaches described above yielded more discriminating results. First, we examined ACE-mediated presentation of the constructs in the presence of increasing doses of purified 2.43, a monoclonal antibody directed to the β subunit of CD8. Although the results were somewhat variable between experiments, this antibody will completely block CTL recognition at doses of 5–10 μg/ml. As shown in Fig. 5, susceptibility to the blocking antibodies across the panel of constructs precisely mirrored the in vitro transport capability of the peptides. Presentation of TG, which exhibited no detectable transport in streptolysin O–permeabilized cells, was severely inhibited at 1 μg/ml of the antibody. This concentration had a lesser, but still significant, blocking effect on TP presentation, a poorly transported substrate. The very well transported substrates, TL and TM, showed only slightly suboptimal presentation levels under this condition. This pattern of differential susceptibility was maintained at decreasing antibody doses. Even at a concentration of 0.1 μg/ml, perhaps one-tenth the concentration of antibody required to block optimally expressed peptide complexes, the poorly transported TG suboptimally sensitized target cells for lysis. Therefore, under conditions of limited T cell stimulation, biochemically established rules of transport capability also appear to apply within the intact cell.


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-rescued presentation under conditions of limited T cell  receptor stimulation. L-Kd cells were coinfected for 4 h with 5 PFU of  the indicated vac alone and 5 PFU ACE vac. 51Cr-labeled target cells  were then assessed for recognition by NP147–155-specific CTLs with or  without the indicated concentrations of anti-CD8 antibody 2.43. CTLs  were used at a constant effector/target ratio of 10:1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: ACE-rescued presentation under conditions of limited T cell receptor stimulation. L-Kd cells were coinfected for 4 h with 5 PFU of the indicated vac alone and 5 PFU ACE vac. 51Cr-labeled target cells were then assessed for recognition by NP147–155-specific CTLs with or without the indicated concentrations of anti-CD8 antibody 2.43. CTLs were used at a constant effector/target ratio of 10:1.
Mentions: When we examined presentation of the epitope from the various TX contexts following the addition of brefeldin A at numerous time points after infection, we failed to distinguish presentation differences between them (data not shown). However, the two other approaches described above yielded more discriminating results. First, we examined ACE-mediated presentation of the constructs in the presence of increasing doses of purified 2.43, a monoclonal antibody directed to the β subunit of CD8. Although the results were somewhat variable between experiments, this antibody will completely block CTL recognition at doses of 5–10 μg/ml. As shown in Fig. 5, susceptibility to the blocking antibodies across the panel of constructs precisely mirrored the in vitro transport capability of the peptides. Presentation of TG, which exhibited no detectable transport in streptolysin O–permeabilized cells, was severely inhibited at 1 μg/ml of the antibody. This concentration had a lesser, but still significant, blocking effect on TP presentation, a poorly transported substrate. The very well transported substrates, TL and TM, showed only slightly suboptimal presentation levels under this condition. This pattern of differential susceptibility was maintained at decreasing antibody doses. Even at a concentration of 0.1 μg/ml, perhaps one-tenth the concentration of antibody required to block optimally expressed peptide complexes, the poorly transported TG suboptimally sensitized target cells for lysis. Therefore, under conditions of limited T cell stimulation, biochemically established rules of transport capability also appear to apply within the intact cell.

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