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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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In vitro transport of synthetic versions of the 147-155TX  constructs. RMA cells were permeabilized with streptolysin O (2 U/ml)  and incubated with radioiodinated reporter peptide (amino acid sequence  TVNKTERAY) plus the indicated dilutions of test peptides. Reporter  peptide transport in TAP-minus RMA-S cells was always assessed as a  negative control and typically yielded counts <2% of those seen with  RMA + reporter peptide alone. Samples were done singly except for  RMA cells with no competitor, done in duplicate. The experiment was  repeated four times, all yielding similar results.
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Figure 3: In vitro transport of synthetic versions of the 147-155TX constructs. RMA cells were permeabilized with streptolysin O (2 U/ml) and incubated with radioiodinated reporter peptide (amino acid sequence TVNKTERAY) plus the indicated dilutions of test peptides. Reporter peptide transport in TAP-minus RMA-S cells was always assessed as a negative control and typically yielded counts <2% of those seen with RMA + reporter peptide alone. Samples were done singly except for RMA cells with no competitor, done in duplicate. The experiment was repeated four times, all yielding similar results.

Mentions: Because each construct sensitized target cells for recognition by CTLs, we considered the possibility that these substrates do not differ in their transport capabilities despite the predicted influence of the COOH-terminal replacements (15, 17). Synthetic versions of selected constructs predicted to be either transport-permissive (147–155TL, –TM) or -nonpermissive (147–155TP, –TE, –TG) were tested for their ability to inhibit TAP-dependent transport of the radiolabeled reporter peptide, TVNKTERAY (17), in streptolysin O–permeabilized RMA cells (Fig. 3). TL and TM were both well-transported, with IC50 values of ∼10 μM. TP was transported at a rate at least a log below that of the well-transported substrates, with inhibition of reporter peptide transport occurring only at substrate concentrations well above those normally tested in this type of assay. Unexpectedly, we frequently observed an enhancement rather than inhibition of reporter peptide transport in the presence of both TE and TG. We are presently investigating this intriguing phenomenon, but based on this data can only state that neither of these substrates appears to be transported under the conditions of this assay. This property was also exhibited by the unappended minimal epitope, generally in agreement with a previously reported IC50 value of >50 μM (14). Therefore, this panel of substrates exhibits a wide range of in vitro transport capabilities. Note that, although rules regarding the influence of COOH-terminal residues on transport have been established on a limited range of test peptides, predictions of transport capability based on these rules were completely borne out.


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)

In vitro transport of synthetic versions of the 147-155TX  constructs. RMA cells were permeabilized with streptolysin O (2 U/ml)  and incubated with radioiodinated reporter peptide (amino acid sequence  TVNKTERAY) plus the indicated dilutions of test peptides. Reporter  peptide transport in TAP-minus RMA-S cells was always assessed as a  negative control and typically yielded counts <2% of those seen with  RMA + reporter peptide alone. Samples were done singly except for  RMA cells with no competitor, done in duplicate. The experiment was  repeated four times, all yielding similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199128&req=5

Figure 3: In vitro transport of synthetic versions of the 147-155TX constructs. RMA cells were permeabilized with streptolysin O (2 U/ml) and incubated with radioiodinated reporter peptide (amino acid sequence TVNKTERAY) plus the indicated dilutions of test peptides. Reporter peptide transport in TAP-minus RMA-S cells was always assessed as a negative control and typically yielded counts <2% of those seen with RMA + reporter peptide alone. Samples were done singly except for RMA cells with no competitor, done in duplicate. The experiment was repeated four times, all yielding similar results.
Mentions: Because each construct sensitized target cells for recognition by CTLs, we considered the possibility that these substrates do not differ in their transport capabilities despite the predicted influence of the COOH-terminal replacements (15, 17). Synthetic versions of selected constructs predicted to be either transport-permissive (147–155TL, –TM) or -nonpermissive (147–155TP, –TE, –TG) were tested for their ability to inhibit TAP-dependent transport of the radiolabeled reporter peptide, TVNKTERAY (17), in streptolysin O–permeabilized RMA cells (Fig. 3). TL and TM were both well-transported, with IC50 values of ∼10 μM. TP was transported at a rate at least a log below that of the well-transported substrates, with inhibition of reporter peptide transport occurring only at substrate concentrations well above those normally tested in this type of assay. Unexpectedly, we frequently observed an enhancement rather than inhibition of reporter peptide transport in the presence of both TE and TG. We are presently investigating this intriguing phenomenon, but based on this data can only state that neither of these substrates appears to be transported under the conditions of this assay. This property was also exhibited by the unappended minimal epitope, generally in agreement with a previously reported IC50 value of >50 μM (14). Therefore, this panel of substrates exhibits a wide range of in vitro transport capabilities. Note that, although rules regarding the influence of COOH-terminal residues on transport have been established on a limited range of test peptides, predictions of transport capability based on these rules were completely borne out.

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