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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–VSV8 complexes reconstituted in vitro elicit peptide-specific, protective immunity. Mice were immunized twice at weekly intervals with gp96–VSV8 complexes (10 μg or 25 μg liver gp96 complexed with 1–2 ng VSV8 peptide), liver gp96 alone (10 or 25 μg), VSV8  peptide alone (75 ng), or RPMI medium control. gp96–VSV8 complexes  were washed extensively using a minicon 50 to remove unbound peptides. All mice were challenged intraperitoneally with 5,000 live N1 cells  1 wk after the second immunization; survival was monitored.
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Figure 6: gp96–VSV8 complexes reconstituted in vitro elicit peptide-specific, protective immunity. Mice were immunized twice at weekly intervals with gp96–VSV8 complexes (10 μg or 25 μg liver gp96 complexed with 1–2 ng VSV8 peptide), liver gp96 alone (10 or 25 μg), VSV8 peptide alone (75 ng), or RPMI medium control. gp96–VSV8 complexes were washed extensively using a minicon 50 to remove unbound peptides. All mice were challenged intraperitoneally with 5,000 live N1 cells 1 wk after the second immunization; survival was monitored.

Mentions: These observations demonstrate that the HSPs gp96 and hsp70 possess an adjuvant activity effective for eliciting a CD8+ T cell response. The efficacy of HSP–peptide vaccination in eliciting tumor rejection was tested in an artificial model, because the identity of peptides that can elicit rejection of natural tumors is yet unknown. Tumor rejection studies were carried out using the N1 tumor, which has been derived by transfection of the EL4 lymphoma of the C57BL/6 mouse with the gene encoding the NP of the VSV. Therefore, VSV–NP is a model tumor rejection antigen for this tumor (16). (In the past, we have actively refrained from using tumor models in which foreign genes have been transfected into tumors, as they reveal little about tumor immunity. However, this model is used in the present study to measure an antigen-specific T cell response in vivo.) Thus, gp96 molecules obtained from normal livers of C57BL/6 mice were complexed with the VSV8 known to bind Kb. C57BL/6 mice were immunized with such reconstituted complexes, or with VSV8 alone, or with liver gp96 alone, and were challenged with N1 cells. Survival of mice was monitored (Fig. 6). It was observed that there was no difference in the survival kinetics of unimmunized mice and mice immunized with liver gp96 or the VSV8 alone: all mice died within 30–50 d of tumor challenge. In contrast, 8 of 10 mice immunized with gp96–VSV8 complexes reconstituted in vitro, survived beyond 100 d after tumor challenge. Spleens of the immunized mice were also tested for antigen-specific CTL response to the VSV8 epitope. It was observed that mice immunized with the gp96–VSV8 complex generated effective antigen-specific, CD8+ CTL response, whereas mice immunized with gp96 alone, or the VSV8 alone, did not (data not shown). These results indicate that the peptides complexed with gp96 in vitro elicit tumor immunity in a manner consistent with the gp96– peptide complexes generated in vivo. Similar antitumor activity has been shown for hsp70–peptide complexes (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–VSV8 complexes reconstituted in vitro elicit peptide-specific, protective immunity. Mice were immunized twice at weekly intervals with gp96–VSV8 complexes (10 μg or 25 μg liver gp96 complexed with 1–2 ng VSV8 peptide), liver gp96 alone (10 or 25 μg), VSV8  peptide alone (75 ng), or RPMI medium control. gp96–VSV8 complexes  were washed extensively using a minicon 50 to remove unbound peptides. All mice were challenged intraperitoneally with 5,000 live N1 cells  1 wk after the second immunization; survival was monitored.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199095&req=5

Figure 6: gp96–VSV8 complexes reconstituted in vitro elicit peptide-specific, protective immunity. Mice were immunized twice at weekly intervals with gp96–VSV8 complexes (10 μg or 25 μg liver gp96 complexed with 1–2 ng VSV8 peptide), liver gp96 alone (10 or 25 μg), VSV8 peptide alone (75 ng), or RPMI medium control. gp96–VSV8 complexes were washed extensively using a minicon 50 to remove unbound peptides. All mice were challenged intraperitoneally with 5,000 live N1 cells 1 wk after the second immunization; survival was monitored.
Mentions: These observations demonstrate that the HSPs gp96 and hsp70 possess an adjuvant activity effective for eliciting a CD8+ T cell response. The efficacy of HSP–peptide vaccination in eliciting tumor rejection was tested in an artificial model, because the identity of peptides that can elicit rejection of natural tumors is yet unknown. Tumor rejection studies were carried out using the N1 tumor, which has been derived by transfection of the EL4 lymphoma of the C57BL/6 mouse with the gene encoding the NP of the VSV. Therefore, VSV–NP is a model tumor rejection antigen for this tumor (16). (In the past, we have actively refrained from using tumor models in which foreign genes have been transfected into tumors, as they reveal little about tumor immunity. However, this model is used in the present study to measure an antigen-specific T cell response in vivo.) Thus, gp96 molecules obtained from normal livers of C57BL/6 mice were complexed with the VSV8 known to bind Kb. C57BL/6 mice were immunized with such reconstituted complexes, or with VSV8 alone, or with liver gp96 alone, and were challenged with N1 cells. Survival of mice was monitored (Fig. 6). It was observed that there was no difference in the survival kinetics of unimmunized mice and mice immunized with liver gp96 or the VSV8 alone: all mice died within 30–50 d of tumor challenge. In contrast, 8 of 10 mice immunized with gp96–VSV8 complexes reconstituted in vitro, survived beyond 100 d after tumor challenge. Spleens of the immunized mice were also tested for antigen-specific CTL response to the VSV8 epitope. It was observed that mice immunized with the gp96–VSV8 complex generated effective antigen-specific, CD8+ CTL response, whereas mice immunized with gp96 alone, or the VSV8 alone, did not (data not shown). These results indicate that the peptides complexed with gp96 in vitro elicit tumor immunity in a manner consistent with the gp96– peptide complexes generated in vivo. Similar antitumor activity has been shown for hsp70–peptide complexes (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