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Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96.

Arnold D, Wahl C, Faath S, Rammensee HG, Schild H - J. Exp. Med. (1997)

Bottom Line: This ability is based on peptides associated with gp96.We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent.These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany.

ABSTRACT
The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

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Induction of β-gal–specific CTL responses using gp96 molecules isolated from P815, P13.1, R/gal, and RS/gal cells. (A) BALB/c  mice were immunized with gp96 molecules isolated from P815 cells (open  triangles), P13.1 cells (closed triangles), or with PBS (closed circles). Spleen  cells were stimulated in vitro with the β-gal–derived peptide TPHPARIGL and the CTL activity was tested on P13.1 cells at the indicated  E/T ratio or on P815 incubated with different concentrations of the β-gal  peptide at an E/T ratio of 3:1. (B) BALB/c mice were immunized with  gp96 molecules isolated from RS/gal cells (open triangles), R/gal cells  (closed triangles), or PBS (closed circles). Spleen cells were stimulated and  CTL activity was determined as described in A.
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Figure 2: Induction of β-gal–specific CTL responses using gp96 molecules isolated from P815, P13.1, R/gal, and RS/gal cells. (A) BALB/c mice were immunized with gp96 molecules isolated from P815 cells (open triangles), P13.1 cells (closed triangles), or with PBS (closed circles). Spleen cells were stimulated in vitro with the β-gal–derived peptide TPHPARIGL and the CTL activity was tested on P13.1 cells at the indicated E/T ratio or on P815 incubated with different concentrations of the β-gal peptide at an E/T ratio of 3:1. (B) BALB/c mice were immunized with gp96 molecules isolated from RS/gal cells (open triangles), R/gal cells (closed triangles), or PBS (closed circles). Spleen cells were stimulated and CTL activity was determined as described in A.

Mentions: To test the hypothesis that peptide binding to gp96 occurs inside the ER we transfected RMA and RMA-S cells with E. coli–derived β-gal resulting in cytosolic expression of the protein (12). β-gal–transfected RMA and RMA-S cell clones, called R/gal and RS/gal and expressing similar levels of the protein were selected (Fig. 1) and expanded for the purification of gp96 according to standard protocols (4). BALB/c mice were immunized with these gp96 preparations and as a control with gp96 from P815 or P13.1, the β-gal transfectant of P815. As shown in Fig. 2 A, only the immunization with gp96 from P13.1 but not P815 induces a CTL response against P13.1 or peptide-incubated P815 cells, underlining the specificity of the gp96 immunization. As shown in Fig. 2 B, the induction of β-gal–specific CTL requires the presence of functional TAP molecules, because only the gp96 preparation from R/gal but not RS/gal cells is able to induce CTL recognizing P13.1 or β-gal peptide-incubated P815 cells. This experiment clearly shows that β-gal–derived peptides are loaded onto gp96 molecules inside the ER, as this process is not observed in cells with a TAP defect. These results were reproduced in three independent experiments with a gp96 concentration (30 μg), which is in threefold excess of the amount required for optimal tumor protection (2) and CTL induction, as determined by titrations of gp96 over a range from 2 to 30 μg (data not shown).


Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96.

Arnold D, Wahl C, Faath S, Rammensee HG, Schild H - J. Exp. Med. (1997)

Induction of β-gal–specific CTL responses using gp96 molecules isolated from P815, P13.1, R/gal, and RS/gal cells. (A) BALB/c  mice were immunized with gp96 molecules isolated from P815 cells (open  triangles), P13.1 cells (closed triangles), or with PBS (closed circles). Spleen  cells were stimulated in vitro with the β-gal–derived peptide TPHPARIGL and the CTL activity was tested on P13.1 cells at the indicated  E/T ratio or on P815 incubated with different concentrations of the β-gal  peptide at an E/T ratio of 3:1. (B) BALB/c mice were immunized with  gp96 molecules isolated from RS/gal cells (open triangles), R/gal cells  (closed triangles), or PBS (closed circles). Spleen cells were stimulated and  CTL activity was determined as described in A.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199000&req=5

Figure 2: Induction of β-gal–specific CTL responses using gp96 molecules isolated from P815, P13.1, R/gal, and RS/gal cells. (A) BALB/c mice were immunized with gp96 molecules isolated from P815 cells (open triangles), P13.1 cells (closed triangles), or with PBS (closed circles). Spleen cells were stimulated in vitro with the β-gal–derived peptide TPHPARIGL and the CTL activity was tested on P13.1 cells at the indicated E/T ratio or on P815 incubated with different concentrations of the β-gal peptide at an E/T ratio of 3:1. (B) BALB/c mice were immunized with gp96 molecules isolated from RS/gal cells (open triangles), R/gal cells (closed triangles), or PBS (closed circles). Spleen cells were stimulated and CTL activity was determined as described in A.
Mentions: To test the hypothesis that peptide binding to gp96 occurs inside the ER we transfected RMA and RMA-S cells with E. coli–derived β-gal resulting in cytosolic expression of the protein (12). β-gal–transfected RMA and RMA-S cell clones, called R/gal and RS/gal and expressing similar levels of the protein were selected (Fig. 1) and expanded for the purification of gp96 according to standard protocols (4). BALB/c mice were immunized with these gp96 preparations and as a control with gp96 from P815 or P13.1, the β-gal transfectant of P815. As shown in Fig. 2 A, only the immunization with gp96 from P13.1 but not P815 induces a CTL response against P13.1 or peptide-incubated P815 cells, underlining the specificity of the gp96 immunization. As shown in Fig. 2 B, the induction of β-gal–specific CTL requires the presence of functional TAP molecules, because only the gp96 preparation from R/gal but not RS/gal cells is able to induce CTL recognizing P13.1 or β-gal peptide-incubated P815 cells. This experiment clearly shows that β-gal–derived peptides are loaded onto gp96 molecules inside the ER, as this process is not observed in cells with a TAP defect. These results were reproduced in three independent experiments with a gp96 concentration (30 μg), which is in threefold excess of the amount required for optimal tumor protection (2) and CTL induction, as determined by titrations of gp96 over a range from 2 to 30 μg (data not shown).

Bottom Line: This ability is based on peptides associated with gp96.We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent.These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany.

ABSTRACT
The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

Show MeSH
Related in: MedlinePlus