By
From the Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, Connecticut 06030
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.
Immunization with heat shock protein (HSP)1 preparations isolated from cancer cells or virus-infected cells has
been reported to elicit protective antitumor or antiviral cellular immune response (1). This paradigm has also been
substantiated in other antigenic systems, such that gp96
HSP preparations isolated from a cell expressing a transfected cytosolic protein can immunize and elicit specific
CTLs against that antigen (9). Similarly, gp96 preparations isolated from cells expressing a given set of minor H antigens can be used to immunize and elicit CTL response
against the minor antigens expressed by the cells that were
the source of the immunizing gp96 preparation (9). HSPs
are not polymorphic molecules and do not differ in their
primary structure among normal tissues and cancers, or
among normal and virus-infected cells. In this light, the remarkably general immunizing ability of HSP preparations has been explained on the basis of the suggestion that the
HSP molecules are associated with peptides generated in
the cells from which the HSPs are isolated (10). Peptides
associated with gp96 and hsp70 have since been demonstrated (6, 11) and it has been shown that dissociation of the
HSP-bound peptides leads to abrogation of immunogenicity
of the HSP preparation (6). Confirmation of these results
has also been obtained in a viral system, as a recent study has
demonstrated that gp96 preparations isolated from vesicular
stomatitis virus (VSV)-infected cells contain VSV-derived
peptides (12). It has been suggested that cytosolic and endoplasmic reticular HSPs chaperone antigenic peptides during antigen processing and presentation by MHC class I
molecules (13). The mechanism by which such noncovalent HSP-peptide complexes elicit protective cellular immune
responses has recently been elucidated (14, 15).
The HSP-peptide interaction is at the center of this
newly emerging immunological paradigm. In this report,
we demonstrate that HSP-peptide complexes can also be generated in vitro and that the biological activity of these
complexes is comparable to that of HSP-peptide complexes
generated in vivo. Further, the HSP-peptide complexes reconstituted in vitro elicit immunity by a mechanism apparently identical to that implicated in the immunogenicity of
the complexes generated in vivo.
Mice and Cell Lines.
Female C57BL/6 (6-8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). EL4 cells
are a thymoma of C57BL/6 origin. N1 is a clone of EL4 transfected with the nucleocapsid gene of VSV (16). VSV-specific
CTLs were derived from mice that were immunized with irradiated (7,500 rads) N1 cells. 7 d after immunization, splenocytes (8 × 106) from immune mice were cultured with irradiated N1 cells (5 × 104) in 24-well plates. CTLs were restimulated every 7 d. Pristane- induced peritoneal exudate cells were used for macrophage enriched population.
Purification of HSPs.
gp96 was purified from C57BL/6 liver
cells, as described (2). In brief, 15 livers were homogenized in 40 ml of hypotonic buffer (30 mM NaHCO3, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.1) by a tissue tearor, and a 100,000 g supernatant was obtained. The supernatant was fractionated by 50-
70% ammonium sulfate precipitation, applied to a concanavalin
A-agarose column, and glycoproteins were eluted by 10% HSP-Peptide Binding.
gp96 and 125I-labeled peptides (synthesized by Bio-Synthesis, Inc., Lewisville, TX), were mixed in the
quantities indicated, and incubated for 10 min at the indicated
temperatures in a binding buffer (20 mM Hepes, pH 7.2, 20 mM
NaCl, and 2 mM MgCl2). The samples were then incubated for
30 min at room temperature. Alternatively, gp96 and peptides
were coincubated in sodium phosphate buffer at 25 or 50°C, as
indicated, for 10 min at various salt concentrations, followed by
incubation at room temperature for 30 min. In the case of hsp70,
high temperatures and high salt concentrations were unnecessary;
hsp70 and peptides were coincubated at 37°C in sodium phosphate buffer containing 1 mM ADP and 1 mM MgCl2. Free peptide was removed completely using a microcon 50 (Amicon, Inc.,
Beverly, MA). The removal of free peptides was monitored by
electrophoretic analysis of the labeling mixture, followed by
quantitative autoradiography; if peptides were not removed, they
were visible on the dye front. Samples were also analyzed by silver staining or immunoblotting with anti-gp96 antibody (anti-GRP94, SPA-850, clone 9G10; NeoMarkers, Fremont, CA) or
anti-hsp70 antibody (clone BRM22 from NeoMarkers). Peptide
quantification was determined by densitometry using the Quantity 1 (version 2.2) program with the PDI Discovery series system
(Sun Microsystems).
Tumor Rejection Assay.
Mice were injected subcutaneously
with 10 µg or 25 µg reconstituted HSP-peptide complexes, or
gp96 alone, peptide alone (75 nM), or buffer twice at weekly intervals. Mice were challenged intraperitoneally with 5,000 live
N1 tumor cells 7 d after the second immunization.
CTL Assay.
Spleen cells (8 × 106/well) from immunized
mice (day 96) were cultured in mixed lymphocyte tumor culture
with 7,500 rads irradiated antigen-positive cells or cognate peptide-pulsed cell (5 × 104/well) in 24-well plates. After 5 d, mixed
lymphocyte tumor cultures were tested for cytotoxicity in a chromium release assay.
TNF- The ability of gp96 molecules to bind peptides in vitro was analyzed using an electrophoretic assay.
The rationale for the use of this assay was as follows: it had
been demonstrated earlier that gp96 preparations obtained
from preparative sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) could still be used to elicit
limited but significant tumor-specific immunity (2). This
observation seen in the context of subsequent studies, which suggested that gp96 preparations are immunogenic
because of association of gp96 with antigenic peptides (11,
12), indicated that gp96-peptide interaction would be expected to be stable under conditions of SDS-PAGE.
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).
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.
Reconstitution of peptides with hsp70 molecules was
observed to require neither a heating and cooling cycle,
nor exposure to high salt concentrations. Incubation of apparently homogeneous preparations of hsp70 with radiolabeled peptide A in sodium phosphate buffer containing 1 mM ADP and 1 mM MgCl2 at 37°C was found to be sufficient to generate SDS-stable hsp70-peptide complexes as judged by autoradiography (see Fig. 5 A).
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.
The specificity of binding of HSPs with the exogenous
peptide was demonstrated in the following additional ways,
as shown here for gp96, but also observed for hsp70. (a) Inclusion of BSA or OVA in gp96-peptide A binding reaction had no influence on gp96-peptide A binding (Fig. 3
B); (b) a number of proteins, i.e., The quantity of peptide bound to the HSPs in vitro was
determined. The specific radioactivity of the peptides (cpm/
mol of peptide) was measured; using this number, the
number of moles of peptides bound to a given quantity of
gp96 were determined by measuring the cpm in the HSP
band after autoradiography, by cutting out the band and
counting it in a gp96-peptide complexes and hsp70-peptide complexes generated in vitro were tested in a variety of models
for their ability to elicit CTLs and tumor immunity. For
generation of CTLs, seven model peptides, which bind to
different MHC class I alleles, were tested. These derive
from OVA (Kb), SV40 T antigen (Db), NP antigen of influenza virus (Db and Kb), NP antigen of VSV (Kb), and
A number of parameters of the adjuvant activity of gp96
and hsp70 were tested. The possibility that complexing of
HSPs with peptides is unnecessary and HSP-peptide mixtures (instead of complexes) may be equally immunogenic
was tested. It was observed that, similar to the results of immunization with HSPs or peptides alone, mixtures of HSPs
and peptides were consistently nonimmunogenic. Similarly, when mice were immunized with HSPs on one flank
and peptides on the other, no CTL response was detected
(data not shown).
The possibility that immunization with peptides mixed
or complexed with any large molecule, particularly other
carrier molecules, might also elicit a potent and specific
CTL response was investigated. The VSV9 peptide (Tyr-
VSV8) was mixed with a traditional carrier protein, mouse
serum albumin, under conditions that facilitate binding
with gp96 and hsp70. The complexed material was analyzed by SDS-PAGE, and mouse serum albumin, like gp96
and hsp70, was found to form an SDS-resistant complex
with the peptide (Fig. 5 A). Mice were immunized with
mouse serum albumin-VSV9 complex, and were tested for
CTL response as described previously. No CTL response
was detected (Fig. 5 B). On the other hand, Gp96-VSV9 and hsp70-VSV9 complexes elicited significant specific
CTL responses (Fig. 5 B).
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).
The mechanism
whereby immunization with gp96-peptide complexes generated in vivo leads to a protective CTL response has been elucidated (15). It has been shown that gp96-peptide complexes are taken up by macrophages and the chaperoned
peptides are re-presented by the MHC class I molecules of
the macrophage through a novel pathway. The immunological activity of the gp96-peptide complexes generated in
vitro was tested in this assay. gp96 preparations were reconstituted with the VSV8 peptide at different temperatures and the resulting complexes were used to pulse pristane-induced macrophages of C57BL/6 mice in vitro. The pulsed
macrophages were tested for their ability to stimulate anti-VSV CTLs, as measured by the secretion of TNF-
The studies described here indicate that HSP-peptide complexes can be reconstituted in vitro and that, by all parameters tested, such complexes show immunological activity
similar to the HSP-peptide complexes generated in vivo.
The results also show significant differences between gp96
and hsp70 with respect to the conditions in vitro, under
which they bind peptides. These differences presumably reflect the fact that although hsp70-ATP interaction plays a
crucial role in hsp70-peptide interaction in vivo, the identity of the corresponding ligand for gp96 is presently unknown. In contrast with the situation with hsp70, gp96-ATP
interaction does not strip gp96 of its associated peptides
(data not shown), even though gp96, like hsp70, is an
ATP-binding protein and is an ATPase (11). Exposure to
high temperature and high salt apparently causes the gp96
molecule to assume an open conformation, which permits
dissociation from and association with exogenous peptides.
The identity of the ligands that catalyze this process in vivo
would be of interest in this regard.
The observations reported here have several implications.
First, they support the hypothesis that immunogenicity of
tumor-derived gp96 preparations results from a physical association of gp96 with antigenic peptides. The HSP-peptide complex elicits immunity under conditions in which
the HSP molecules alone, or the peptides alone, do not.
Second, these observations show that one does not have to
rely on HSP-peptide complexes generated in vivo to elicit immunity; instead, such complexes can be generated reproducibly in vitro, provided the identity of the immunogenic
peptides is known. A variety of peptides of different lengths,
compositions, and hydrophobicity can bind the HSPs, suggesting that the nature of an epitope is not a limiting factor
in its suitability as a vaccine in the form of a HSP-peptide
complex. The ability of the gp96 to bind peptides in vitro
has also been independently demonstrated recently (20).
The quantity of peptide that is required to be conjugated to
the HSPs is extremely small and 1-2 ng of peptides complexed to the HSPs elicit potent cellular immune response. At first sight, this quantity may appear to be unrealistically small; however, when it is considered that the peptides
chaperoned by the HSPs are targeted specifically to the
professional antigen-presenting cells (15, 21), 1-2 ng or ~6 × 1011 molecules of specific peptide targeted to the relevant antigen-presenting cells are actually a large number, as
argued in more detail elsewhere (22). This observation has
significant implications for vaccination against infectious
diseases in which the protective epitopes are known, and
for any cancers, such as those of viral etiology, that may
share antigenic epitopes.
Essentially, these results show that HSPs are adjuvants.
This adjuvanticity has a number of unique characteristics:
in contrast with other nonlive adjuvants, the adjuvanticity
of HSPs generates MHC class I-restricted T cell responses.
No serological antipeptide response has ever been detected
among the tens of immunized mice tested (data not shown).
The quantitative requirements of antigens administered
with HSPs are log scales lower than corresponding requirements for other adjuvants. Finally, HSPs are the first adjuvants of mammalian origin. We have suggested previously
that the immunogenicity of HSP-peptide complexes may
reflect the role in vivo of such complexes in priming of cellular immune responses (23). In this view, the observed adjuvanticity of HSPs is simply a reflection of the natural role
of HSPs in vivo.
The structural basis of the ability of gp96 molecules to
bind a variety of peptides is presently unclear and requires
further study. Obviously, there are certain rules for the
HSP-peptide interaction as seen in the observation that
peptides differ in their ability to compete with a given peptide for binding to gp96 (Fig. 3). However, the studies carried out here are not of a broad enough scope to permit
elucidation of these rules. Broadly speaking, HSP-peptide
interaction is reminiscent of MHC-peptide interaction, which was equally mysterious as to its structural basis until the rules of interaction were identified (24). The MHC and
the HSPs share a number of crucial properties, such as the
ability to bind peptides, a ubiquitous tissue distribution,
high degree of phylogenetic conservation, inducibility of
the respective genes by IFN-
-methylmannoside. The eluate was applied to a DEAE-agarose column,
equilibrated with 0.3 M NaCl, and was eluted with 0.7 M NaCl.
Hsp70 was purified as described by Peng et al. (17).
Bioassay.
Macrophages (1.5 × 104) and VSV-specific
CTLs (5 × 104) were cultured with serially diluted reconstituted
VSV-gp96 complexes for 24 h at 37°C. Supernatants were collected and assayed for TNF-
production in a cytotoxicity assay
as described (15).
Exchange of Peptides Naturally Bound to HSPs with Exogenous Peptides.
Fig. 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).
[View Larger Version of this Image (36K GIF file)]
Fig. 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).
[View Larger Version of this Image (23K GIF file)]
Fig. 5.
Chaperoning of
peptides by HSPs is required for
generation of an effective CD8+
T cell response. gp96, hsp70, or
mouse serum albumin (MSA)
were complexed with radiolabeled VSV9 and analyzed by (A)
SDS-PAGE followed by Coomassie blue staining and autoradiography. In addition, mice
were immunized (B) with peptides complexed or simply mixed with each of the proteins. Splenocytes of these mice were
tested for induction of CD8+ T
lymphocytes, as described in legend to Fig. 4. N1 (closed circle) and EL4 (open circle) were used as
targets.
[View Larger Versions of these Images (12 + 48K GIF file)]
Fig. 4.
Hsp-peptide complexes reconstituted in vitro prime mice for CD8+ T cell response. Mice were immunized with HSPs alone (20-50 µg), peptides alone (10 µg), or HSP-peptide complexes (20-50 µg), as indicated. 1 wk after the last immunization, spleens were removed and stimulated with
the cognate peptide or with cells transfected with the gene encoding the relevant antigen. The lymphocyte cultures were tested for their ability to lyse
cells transfected with the antigen of interest (closed circle) and the nontransfected parental line (open circle). For the top panel, HSPs alone were tested for
their immunizing ability in each antigenic system and were found to be consistently negative. The CTL responses were tested in many but not all systems
and where tested, were found to be MHC class I and CD8 restricted.
[View Larger Version of this Image (20K GIF file)]
Fig. 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., -2 macroglobulin,
-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.
[View Larger Version of this Image (45K GIF file)]
-2 macroglobulin,
-galactosidase, fructose-6-phosphate kinase, OVA, pyruvate kinase,
fumarase, and triosephosphate isomerase were tested for their
ability to bind peptide A and were observed to not bind it
(Fig. 3 C); and (c) it was demonstrated that only the intact
gp96 and not any of its various degradation products could
bind peptide A (Fig. 3 C).
counter. This calculation revealed that
under the conditions tested, and assuming a stoichiometry of one peptide per HSP molecule, ~1% of HSP molecules
were loaded with the exogenous peptide. The assumption
of a 1:1 stoichiometry between HSP and peptides was
made on the basis of the recent demonstration of a single
peptide-binding pocket in a bacterial hsp70 molecule (19).
-galactosidase (Ld). The peptides were complexed with
gp96, hsp70, or both and mice of the appropriate haplotype
(b or d) were immunized twice at weekly intervals, with
the peptides alone (10 µg peptide in PBS), the uncomplexed HSPs alone (20-50 µg HSP in PBS), or the HSP- peptide complexes (20-50 µg HSP complexed with ~2 ng
peptide, determined as described in the previous section).
Spleen cells from the immunized mice were put in culture
and were stimulated with the cognate peptide and tested
for cytotoxic activity on target cells pulsed or unpulsed
with relevant peptides. It was observed (Fig. 4) that T cells
obtained from mice immunized with peptides alone or
with gp96 or hsp70 alone showed no cytotoxic activity,
whereas T cells obtained from mice immunized with HSP- peptide complexes showed significant and consistent peptide-specific CTL activity. The precise MHC class I-binding peptides were used in these studies. To test whether
larger peptides than the exact epitopes could be complexed
with HSPs and used to immunize successfully, a 19-mer
precursor of Kb-binding VSV8 (VSV19) was complexed
with hsp70 and gp96 and the complexes used to immunize.
The HSP-VSV19 complexes were found to be as effective
at eliciting antigen-specific CTL response as the HSP-
VSV8 complexes were (Fig. 4).
Fig. 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.
[View Larger Version of this Image (16K GIF file)]
by the
CTLs (Fig. 7). It was observed that the macrophage pulsed
with complexes reconstituted at 60°C were effective in this
re-presentation assay, whereas those reconstituted at 37, 80, or 98°C were not. As we have shown previously (15), the
quantity of VSV8 complexed with gp96 in these experiments is ~2 log scales lower than that necessary for direct
charging of the empty surface MHC class I molecules by
the VSV8 peptides. Data in Fig. 7 show that the gp96-peptide complexes reconstituted in vitro appear to be re-presented by the antigen-presenting cells in the same manner
as shown previously (15) for the natural HSP-peptide complexes.
Fig. 7.
VSV8 peptide chaperoned by gp96 is re-presented
by macrophage. Pristane-induced
peritoneal exudate cells (104) and
VSV peptide-specific CTL (5 × 104) were cocultured with gp96-
VSV8 complexes (3-20 µg/ml)
reconstituted at 37, 60, 85, or
98°C in a 96-well U-bottomed
plate at 37°C. After 24 h, supernatants were collected and assayed
for TNF- production in a cytotoxicity assay as described (15).
[View Larger Version of this Image (20K GIF file)]
(25) and, finally, the ability
to prime CTL responses against the peptides chaperoned by
them. These considerations led us in the past (26) to suggest a phylogenetic relationship between the MHC and the
HSPs, and a number of recent observations (19, 27)
have not been inconsistent with that suggestion. The association of peptides with HSPs of the cytosol (hsp70 and hsp90) and the endoplasmic reticulum (gp96) had also led
us to suggest that HSPs constitute a relay line of molecules
that chaperones the peptides and ultimately delivers them
to the MHC class I molecules (23). Therefore, the HSPs
were suggested to be accessories to antigen presentation by
MHC class I molecules. Our recent results, which show
that peptides precursors to the MHC class I-binding epitopes
are found in specific association with hsp70, hsp90, and
gp96 (Ishii et al., manuscript submitted for publication), are
in accord with our suggestion. The recent demonstration
by Lammert et al. (29) that the HSP gp96 acts as a major
peptide acceptor for peptides transported into the lumen of
the endoplasmic reticulum through transport-associated
protein molecules, also supports the relay-line hypothesis.
Address correspondence to Pramod K. Srivastava, PhD, Center for Immunotherapy of Cancer and Infectious Diseases; University of Connecticut School of Medicine, MC1601, Farmington, CT 06030. Phone: 860-679-4444; FAX: 860-679-4365; E-mail: srivastava{at}nso2.uchc.edu. The present address of Zihai Li is Fred Hutchinson Cancer Center, Clinical Research Division, 1124 Columbia, Seattle, WA 98104. The present address of Rajiv Chandawarkar is Department of Surgery, Akron General Medical Center, Akron, OH 44307. The present address of Heiichiro Udono is Department of Parasitology and Immunology, Okayama University School of Medicine, Okayama 700, Japan.
Received for publication 30 June 1997 and in revised form 31 July 1997.
N.E. Blachere and Z. Li contributed equally to this work and are listed in alphabetical order.The authors are grateful to Antoine Menoret and Ping Peng for critical reading of the manuscript.
This work was supported by National Institutes of Health grants CA44786 and CA64394, and a sponsored research agreement with Antigenics, Inc (New York, NY). R. Suto was a postdoctoral fellow of the Cancer Research Institute, New York.
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