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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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Related in: MedlinePlus

Exchange of exogenous and native-bound peptides at high  salt concentrations. gp96 (40 pM) and iodinated synthetic peptide (2 nM,  NH2–Ser–Leu–Ser–Asp–Leu–Arg–Gly–Tyr–Val–Tyr–Gln– Gly– Leu– Lys–  Ser–Gly–Asn–Val–Ser–COOH) were mixed in phosphate buffer in 20 μl  reaction volume and incubated at 25 or 50°C for 10 min. After centrifugation, the mixtures were incubated at 25°C for another 30 min. Samples  were analyzed by SDS-PAGE and staining, followed by autoradiography  of the stained gel (24-h exposure).
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Figure 2: Exchange of exogenous and native-bound peptides at high salt concentrations. gp96 (40 pM) and iodinated synthetic peptide (2 nM, NH2–Ser–Leu–Ser–Asp–Leu–Arg–Gly–Tyr–Val–Tyr–Gln– Gly– Leu– Lys– Ser–Gly–Asn–Val–Ser–COOH) were mixed in phosphate buffer in 20 μl reaction volume and incubated at 25 or 50°C for 10 min. After centrifugation, the mixtures were incubated at 25°C for another 30 min. Samples were analyzed by SDS-PAGE and staining, followed by autoradiography of the stained gel (24-h exposure).

Mentions: The exchange of exogenous and native-bound peptides could also be achieved by incubation of gp96 with exogenous peptides at high salt concentrations. Gp96 preparations were incubated at 25 or 50°C with radio-iodinated peptide VSV19 (extended on both termini of Kb-binding VSV nucleocapsid protein (NP)-derived octamer VSV8) for 10 min in sodium phosphate buffer containing 200 mM, 300 mM, 500 mM, 700 mM, 800 mM, 1 M, 2 M, or 3 M NaCl, followed by 30 min at room temperature. The samples were desalted and analyzed by SDS-PAGE, followed by staining as well as autoradiography. It was observed (Fig. 2) that significant quantities of labeled peptides formed an SDS-resistant association with gp96 after incubation at 2 M or higher NaCl concentration, but not at lower concentrations. In presence of high salt, the extent of association of gp96 with peptides was comparable at the low and high temperatures, whereas at low salt concentrations, gp96–peptide interaction was detected only at the higher temperature. The quantity of gp96 in each lane, as judged by Coomassie blue staining and scanning, was identical.


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)

Exchange of exogenous and native-bound peptides at high  salt concentrations. gp96 (40 pM) and iodinated synthetic peptide (2 nM,  NH2–Ser–Leu–Ser–Asp–Leu–Arg–Gly–Tyr–Val–Tyr–Gln– Gly– Leu– Lys–  Ser–Gly–Asn–Val–Ser–COOH) were mixed in phosphate buffer in 20 μl  reaction volume and incubated at 25 or 50°C for 10 min. After centrifugation, the mixtures were incubated at 25°C for another 30 min. Samples  were analyzed by SDS-PAGE and staining, followed by autoradiography  of the stained gel (24-h exposure).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199095&req=5

Figure 2: Exchange of exogenous and native-bound peptides at high salt concentrations. gp96 (40 pM) and iodinated synthetic peptide (2 nM, NH2–Ser–Leu–Ser–Asp–Leu–Arg–Gly–Tyr–Val–Tyr–Gln– Gly– Leu– Lys– Ser–Gly–Asn–Val–Ser–COOH) were mixed in phosphate buffer in 20 μl reaction volume and incubated at 25 or 50°C for 10 min. After centrifugation, the mixtures were incubated at 25°C for another 30 min. Samples were analyzed by SDS-PAGE and staining, followed by autoradiography of the stained gel (24-h exposure).
Mentions: The exchange of exogenous and native-bound peptides could also be achieved by incubation of gp96 with exogenous peptides at high salt concentrations. Gp96 preparations were incubated at 25 or 50°C with radio-iodinated peptide VSV19 (extended on both termini of Kb-binding VSV nucleocapsid protein (NP)-derived octamer VSV8) for 10 min in sodium phosphate buffer containing 200 mM, 300 mM, 500 mM, 700 mM, 800 mM, 1 M, 2 M, or 3 M NaCl, followed by 30 min at room temperature. The samples were desalted and analyzed by SDS-PAGE, followed by staining as well as autoradiography. It was observed (Fig. 2) that significant quantities of labeled peptides formed an SDS-resistant association with gp96 after incubation at 2 M or higher NaCl concentration, but not at lower concentrations. In presence of high salt, the extent of association of gp96 with peptides was comparable at the low and high temperatures, whereas at low salt concentrations, gp96–peptide interaction was detected only at the higher temperature. The quantity of gp96 in each lane, as judged by Coomassie blue staining and scanning, was identical.

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