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Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity.

Blachere NE, Li Z, Chandawarkar RY, Suto R, Jaikaria NS, Basu S, Udono H, Srivastava PK - J. Exp. Med. (1997)

Bottom Line: The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs.The studies reported here demonstrate that immunogenic HSP-peptide complexes can also be reconstituted in vitro.These observations demonstrate that HSPs are CD8+ T cell response-eliciting adjuvants.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington 06030, USA.

ABSTRACT
Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP-peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP-peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8+ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96- peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8+ T cell response-eliciting adjuvants.

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gp96 binds to peptides in vitro. gp96 (10 pmol) was incubated with increasing concentrations of radioiodinated peptide A (25, 75,  and 125 pmol) for 10 min at different temperatures in 20 μl reaction  buffer, followed by 30 min at room temperature. The reaction was terminated by mixing with sample buffer (0.1% SDS, 20% glycerol, and 5%  bromophenol blue) and analyzed by SDS-PAGE. (A) Autoradiogram after 48-h exposure. (B) Densitometric quantification of results in A. An aliquot of each reaction was analyzed in parallel by SDS-PAGE and silver  staining (C).
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Figure 1: gp96 binds to peptides in vitro. gp96 (10 pmol) was incubated with increasing concentrations of radioiodinated peptide A (25, 75, and 125 pmol) for 10 min at different temperatures in 20 μl reaction buffer, followed by 30 min at room temperature. The reaction was terminated by mixing with sample buffer (0.1% SDS, 20% glycerol, and 5% bromophenol blue) and analyzed by SDS-PAGE. (A) Autoradiogram after 48-h exposure. (B) Densitometric quantification of results in A. An aliquot of each reaction was analyzed in parallel by SDS-PAGE and silver staining (C).

Mentions: The peptide A (KRQIYTDLEMNRLGK) derived from G protein of the VSV was used for the initial studies. This peptide has been shown previously to bind hsp70 molecules in vitro (18). Apparently homogeneous, unlabeled gp96 preparations were incubated at 37°C with iodinated peptide A as described in Materials and Methods. The sample was analyzed by SDS-PAGE with or without additional heating of the sample in SDS-PAGE sample buffer, followed by autoradiography. The expectation from such an experiment is that SDS-resistant binding of unlabeled gp96 to labeled peptide will result in a labeled 96-kD band. However, no binding of gp96 to peptide A is detected under these conditions. The possibility was considered that incubation of gp96 with exogenous peptides at higher temperatures might permit dissociation of naturally bound peptides followed by reannealing of a proportion of exogenously added radiolabeled peptides at lower temperatures. Iodinated preparations of peptide A were incubated with unlabeled gp96 at 4, 25, 37, 60, or 90°C for 10 min and allowed to cool to room temperature for an additional 30 min. The samples were analyzed by SDS-PAGE without further heating and the gels were stained for proteins and autoradiographed. It was observed (Fig. 1) that exogenously added labeled peptide A could associate with gp96 in a temperature-dependent manner with optimal binding at 60°C. Little binding is detected at 4, 25, 37, or 90°C. Although the intensity of label in the gp96 band varies at different temperatures and at different peptide concentrations (Fig. 1, A and B), the quantity of gp96 as detected by silver staining is constant in all lanes (Fig. 1 C). It was also observed that the gp96–peptide binding can be dissociated, if the complexes are heated in a boiling water bath (data not shown).


Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity.

Blachere NE, Li Z, Chandawarkar RY, Suto R, Jaikaria NS, Basu S, Udono H, Srivastava PK - J. Exp. Med. (1997)

gp96 binds to peptides in vitro. gp96 (10 pmol) was incubated with increasing concentrations of radioiodinated peptide A (25, 75,  and 125 pmol) for 10 min at different temperatures in 20 μl reaction  buffer, followed by 30 min at room temperature. The reaction was terminated by mixing with sample buffer (0.1% SDS, 20% glycerol, and 5%  bromophenol blue) and analyzed by SDS-PAGE. (A) Autoradiogram after 48-h exposure. (B) Densitometric quantification of results in A. An aliquot of each reaction was analyzed in parallel by SDS-PAGE and silver  staining (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199095&req=5

Figure 1: gp96 binds to peptides in vitro. gp96 (10 pmol) was incubated with increasing concentrations of radioiodinated peptide A (25, 75, and 125 pmol) for 10 min at different temperatures in 20 μl reaction buffer, followed by 30 min at room temperature. The reaction was terminated by mixing with sample buffer (0.1% SDS, 20% glycerol, and 5% bromophenol blue) and analyzed by SDS-PAGE. (A) Autoradiogram after 48-h exposure. (B) Densitometric quantification of results in A. An aliquot of each reaction was analyzed in parallel by SDS-PAGE and silver staining (C).
Mentions: The peptide A (KRQIYTDLEMNRLGK) derived from G protein of the VSV was used for the initial studies. This peptide has been shown previously to bind hsp70 molecules in vitro (18). Apparently homogeneous, unlabeled gp96 preparations were incubated at 37°C with iodinated peptide A as described in Materials and Methods. The sample was analyzed by SDS-PAGE with or without additional heating of the sample in SDS-PAGE sample buffer, followed by autoradiography. The expectation from such an experiment is that SDS-resistant binding of unlabeled gp96 to labeled peptide will result in a labeled 96-kD band. However, no binding of gp96 to peptide A is detected under these conditions. The possibility was considered that incubation of gp96 with exogenous peptides at higher temperatures might permit dissociation of naturally bound peptides followed by reannealing of a proportion of exogenously added radiolabeled peptides at lower temperatures. Iodinated preparations of peptide A were incubated with unlabeled gp96 at 4, 25, 37, 60, or 90°C for 10 min and allowed to cool to room temperature for an additional 30 min. The samples were analyzed by SDS-PAGE without further heating and the gels were stained for proteins and autoradiographed. It was observed (Fig. 1) that exogenously added labeled peptide A could associate with gp96 in a temperature-dependent manner with optimal binding at 60°C. Little binding is detected at 4, 25, 37, or 90°C. Although the intensity of label in the gp96 band varies at different temperatures and at different peptide concentrations (Fig. 1, A and B), the quantity of gp96 as detected by silver staining is constant in all lanes (Fig. 1 C). It was also observed that the gp96–peptide binding can be dissociated, if the complexes are heated in a boiling water bath (data not shown).

Bottom Line: The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs.The studies reported here demonstrate that immunogenic HSP-peptide complexes can also be reconstituted in vitro.These observations demonstrate that HSPs are CD8+ T cell response-eliciting adjuvants.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington 06030, USA.

ABSTRACT
Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP-peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP-peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8+ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96- peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8+ T cell response-eliciting adjuvants.

Show MeSH
Related in: MedlinePlus