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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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Effect of a panel of thermostable duplex barriers on NP expression. (A) Schematic diagram of the hairpin structures. Arrows indicate  the sites of mismatched bps. (B) L-Kd cells were infected for 1 h with 10  PFU of the indicated vacs, followed by an additional 5 h in the presence  of [35S]methionine. Cells were lysed and protein A–Sepharose–cleared lysates were subjected to immunoprecipitation by NP-specific antibodies.
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Figure 6: Effect of a panel of thermostable duplex barriers on NP expression. (A) Schematic diagram of the hairpin structures. Arrows indicate the sites of mismatched bps. (B) L-Kd cells were infected for 1 h with 10 PFU of the indicated vacs, followed by an additional 5 h in the presence of [35S]methionine. Cells were lysed and protein A–Sepharose–cleared lysates were subjected to immunoprecipitation by NP-specific antibodies.

Mentions: To quantitatively ascertain the effects of these barrier structures on steady state substrate expression levels, the expression of vac-encoded full-length NP was assessed by NP-specific immunoprecipitation of metabolically labeled vac-infected cells. We found that the most thermostable of these, HP20, reduced NP expression to undetectable levels (>100-fold reduction) by this biochemical approach, while HP18 reduced expression by ∼100-fold. Interestingly, the HP16 and HP14 structures showed similar decreases of approximately two- to fivefold (Fig. 6).


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)

Effect of a panel of thermostable duplex barriers on NP expression. (A) Schematic diagram of the hairpin structures. Arrows indicate  the sites of mismatched bps. (B) L-Kd cells were infected for 1 h with 10  PFU of the indicated vacs, followed by an additional 5 h in the presence  of [35S]methionine. Cells were lysed and protein A–Sepharose–cleared lysates were subjected to immunoprecipitation by NP-specific antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effect of a panel of thermostable duplex barriers on NP expression. (A) Schematic diagram of the hairpin structures. Arrows indicate the sites of mismatched bps. (B) L-Kd cells were infected for 1 h with 10 PFU of the indicated vacs, followed by an additional 5 h in the presence of [35S]methionine. Cells were lysed and protein A–Sepharose–cleared lysates were subjected to immunoprecipitation by NP-specific antibodies.
Mentions: To quantitatively ascertain the effects of these barrier structures on steady state substrate expression levels, the expression of vac-encoded full-length NP was assessed by NP-specific immunoprecipitation of metabolically labeled vac-infected cells. We found that the most thermostable of these, HP20, reduced NP expression to undetectable levels (>100-fold reduction) by this biochemical approach, while HP18 reduced expression by ∼100-fold. Interestingly, the HP16 and HP14 structures showed similar decreases of approximately two- to fivefold (Fig. 6).

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