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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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Specificity of peptide binding by gp96. (A) Unlabeled peptides A, B, C, D, and E (0.1 and 10 nM) were used to compete with labeled peptide A (25 pmol) for the binding to gp96 (10 pmol). The sequences of these peptides are described in the text. The binding assay  described in the legend to Fig. 1 was used, except that binding was carried  out at 50°C. (B) Albumin and ovalbumin do not compete with gp96 for  binding to peptide A. gp96 (10 pmol) was incubated with 25-pmol radiolabeled peptide A and analyzed as in the legend to Fig. 1. Albumin and  ovalbumin (10 pmol each) were included in the binding assay. The autoradiogram and the silver stained gel are shown. (C) A partially degraded  preparation of gp96 and a mixture of six purified proteins (i.e., α–2 macroglobulin, β-galactosidase, fructose-6-phosphokinase, pyruvate kinase,  fumarase, and triosephosphate isomerase) were tested for binding to iodinated peptide A. Only the intact gp96 molecule is able to form a stable  complex with radioactive peptide A. None of the other six proteins tested  are able to bind peptide A.
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Figure 3: Specificity of peptide binding by gp96. (A) Unlabeled peptides A, B, C, D, and E (0.1 and 10 nM) were used to compete with labeled peptide A (25 pmol) for the binding to gp96 (10 pmol). The sequences of these peptides are described in the text. The binding assay described in the legend to Fig. 1 was used, except that binding was carried out at 50°C. (B) Albumin and ovalbumin do not compete with gp96 for binding to peptide A. gp96 (10 pmol) was incubated with 25-pmol radiolabeled peptide A and analyzed as in the legend to Fig. 1. Albumin and ovalbumin (10 pmol each) were included in the binding assay. The autoradiogram and the silver stained gel are shown. (C) A partially degraded preparation of gp96 and a mixture of six purified proteins (i.e., α–2 macroglobulin, β-galactosidase, fructose-6-phosphokinase, pyruvate kinase, fumarase, and triosephosphate isomerase) were tested for binding to iodinated peptide A. Only the intact gp96 molecule is able to form a stable complex with radioactive peptide A. None of the other six proteins tested are able to bind peptide A.

Mentions: Binding of gp96 or hsp70 to peptides is not restricted to peptide A and can also be demonstrated for an array of other peptides, such as peptide B, LSSLFRPKRRPIYKS (derived from VSV G protein; reference 18); peptide C, SLSDLRGYVYQGLKSGNVS (derived from VSV nucleoprotein; reference 18); peptide D, IASNENMETMESSTLE (derived from nucleoprotein of influenza virus strain A/PR/8/34); and peptide E, SFIRGTKVSPRGKLST (derived from nucleoprotein of influenza virus A/NY/60/ 68) (data not shown). To evaluate the specificity of binding of peptides to gp96, unlabeled peptides A, B, C, D, and E were tested for their ability to compete with labeled peptide A in the gp96–peptide A binding assay. gp96, 25 pmol radiolabeled peptide A, and 0.1 or 10 nmol unlabeled peptides A, B, C, D, or E were coincubated at 50°C, followed by a 30-min incubation at room temperature. It was observed that all peptides could compete with peptide A in binding to gp96, although with different efficiencies (Fig. 3 A). As expected, higher quantities (10 nmol) of competing unlabeled peptides were more effective in displacing labeled peptide A than the lower quantities in the case of all peptides except peptide E, in which case the competition was already saturating at the lower quantity.


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)

Specificity of peptide binding by gp96. (A) Unlabeled peptides A, B, C, D, and E (0.1 and 10 nM) were used to compete with labeled peptide A (25 pmol) for the binding to gp96 (10 pmol). The sequences of these peptides are described in the text. The binding assay  described in the legend to Fig. 1 was used, except that binding was carried  out at 50°C. (B) Albumin and ovalbumin do not compete with gp96 for  binding to peptide A. gp96 (10 pmol) was incubated with 25-pmol radiolabeled peptide A and analyzed as in the legend to Fig. 1. Albumin and  ovalbumin (10 pmol each) were included in the binding assay. The autoradiogram and the silver stained gel are shown. (C) A partially degraded  preparation of gp96 and a mixture of six purified proteins (i.e., α–2 macroglobulin, β-galactosidase, fructose-6-phosphokinase, pyruvate kinase,  fumarase, and triosephosphate isomerase) were tested for binding to iodinated peptide A. Only the intact gp96 molecule is able to form a stable  complex with radioactive peptide A. None of the other six proteins tested  are able to bind peptide A.
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Figure 3: Specificity of peptide binding by gp96. (A) Unlabeled peptides A, B, C, D, and E (0.1 and 10 nM) were used to compete with labeled peptide A (25 pmol) for the binding to gp96 (10 pmol). The sequences of these peptides are described in the text. The binding assay described in the legend to Fig. 1 was used, except that binding was carried out at 50°C. (B) Albumin and ovalbumin do not compete with gp96 for binding to peptide A. gp96 (10 pmol) was incubated with 25-pmol radiolabeled peptide A and analyzed as in the legend to Fig. 1. Albumin and ovalbumin (10 pmol each) were included in the binding assay. The autoradiogram and the silver stained gel are shown. (C) A partially degraded preparation of gp96 and a mixture of six purified proteins (i.e., α–2 macroglobulin, β-galactosidase, fructose-6-phosphokinase, pyruvate kinase, fumarase, and triosephosphate isomerase) were tested for binding to iodinated peptide A. Only the intact gp96 molecule is able to form a stable complex with radioactive peptide A. None of the other six proteins tested are able to bind peptide A.
Mentions: Binding of gp96 or hsp70 to peptides is not restricted to peptide A and can also be demonstrated for an array of other peptides, such as peptide B, LSSLFRPKRRPIYKS (derived from VSV G protein; reference 18); peptide C, SLSDLRGYVYQGLKSGNVS (derived from VSV nucleoprotein; reference 18); peptide D, IASNENMETMESSTLE (derived from nucleoprotein of influenza virus strain A/PR/8/34); and peptide E, SFIRGTKVSPRGKLST (derived from nucleoprotein of influenza virus A/NY/60/ 68) (data not shown). To evaluate the specificity of binding of peptides to gp96, unlabeled peptides A, B, C, D, and E were tested for their ability to compete with labeled peptide A in the gp96–peptide A binding assay. gp96, 25 pmol radiolabeled peptide A, and 0.1 or 10 nmol unlabeled peptides A, B, C, D, or E were coincubated at 50°C, followed by a 30-min incubation at room temperature. It was observed that all peptides could compete with peptide A in binding to gp96, although with different efficiencies (Fig. 3 A). As expected, higher quantities (10 nmol) of competing unlabeled peptides were more effective in displacing labeled peptide A than the lower quantities in the case of all peptides except peptide E, in which case the competition was already saturating at the lower quantity.

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