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Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
when incubated
with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides,
HSP-mediated secretion of TNF- and IFN- requires cell contact of
the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70
molecules purified from Ag-negative tissue, e.g., negative for
antigenic peptide, are able to activate T cells in vivo, leading to
significant higher frequencies in OVA-specific CD8+ T
cells. In unprimed animals, these T cells lyse OVA-transfected cell
lines and produce TNF- and IFN- after Ag stimulus. Taken together
our data show that, besides the well-established HSP/peptide-specific
CTL induction and activation, a second mechanism exists by which Hsp70
and gp96 molecules activate T cells in vivo and in
vitro. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ T cells play a major role in HSP-mediated tumor regression, whereas immunization with tumor cells also requires CD4+ T cells 5 . The paradigm of HSP immunization has also been extended to other antigenic model systems: gp96 preparations isolated from cells expressing a given set of minor H Ags or expressing a transfected ß-galactosidase (ß-gal) protein 6 can be used to immunize and elicit CTL responses against the minor Ags or ß-gal-derived peptides, respectively. Confirmation of these results has been obtained in an independent antigenic system in which vesicular stomatitis virus (VSV)-infected cells were the source for the purified gp96 molecules 7 . gp96 molecules isolated from VSV-infected cells contained the major H2-Kb binding epitope from the VSV nucleoprotein VSV52–59 (RGYVYQGL). This peptide associates with gp96 in VSV-infected cells regardless of the MHC haplotype of the cell providing a biochemical explanation for the "cross-priming" 8 phenomenon that can be observed after immunization with gp96 preparations.
All three described model systems (minor H, ß-gal, and the VSV-Ags) investigating the function of HSP-mediated CTL induction depend on the presence of HSP-associated peptides. Minor H Ag-negative or uninfected cell lines, as a source of HSP, do not work effectively in these immunization protocols.
In this report, we analyzed a potential capacity of HSP molecules to activate T cells that are not restricted to HSP-bound peptides. We used the well-known OVA-antigenic system and purified Hsp70 and gp96 from the OVA-transfected cell line E.G7 9 . E.G7 cells process and present the H2-Kb binding epitope OVA257–264 (SIINFEKL) 10, 11 that can be detected in in vitro chromium release assays with the help of the H2-Kb/SIINFEKL-specific CTL clone 4G3 12 . In contrast to the described systems that were dependent on HSP-associated antigenic peptides, immunization with HSPs derived from OVA-negative tissue leads to the appearance of H2-Kb/SIINFEKL-specific CTL lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-week-old BALB/c and C57BL/6 mice were obtained from
Charles River WIGA (Sulzfeld, Germany) and maintained in the animal
facilities at the Bernhard-Nocht-Institute. A-20 is a H-2d
B-cell line, EL4 is a H-2b T cell lymphoma, and E.G7 cells
are EL4 cells transfected with the chicken/OVA gene 9 . P13.1 cells
(H-2d) are P815 cells transfected with the ß-gal gene
6 . 4G3 represents a SIINFEKL/H-2b, 0805B a
TPHPARIGL/H-2d-specific CTL clone. The synthetic
SIINFEKL peptide was a kind gift of Prof. H. U. Weltzien
(Max-Planck-Institute for Immunobiology, Freiburg, Germany). The
ß-gal peptide and CTL clone 0805B were provided by Dr. H. G.
Rammensee (Department of Immunology, Institute of Cell Biology,
Eberhard-Karls-University, Tübingen, Germany). All cells were
cultured in RPMI 1640 medium supplemented with 5% FCS, 2ß-ME, and
L-glutamine. In the case of E.G7 250 µg/ml and in the
case of P13.1 cells, 1 mg/ml of G418 was added as a selection
antibiotic. CTL clones were supplemented with 10% FCS and IL-2 (100
U/ml). The CTL line 4G3 was restimulated weekly with irradiated E.G7
cells (7 x 105/ml). In the case of ß-gal-specific
0805B cells, irradiated syngenic spleen cells (106/ml) and
1 µM ß-gal peptide were incubated in 96-well round-bottom culture
dishes (Greiner, Nurtingen, Germany). To induce peritoneal
macrophages, mice were injected i.p. with 500 µl pristane (Sigma,
Deisenhofen, Germany). Peritoneal exudate cells (PEC) were harvested
5–6 days later by rinsing the peritoneum with ice cold medium. Only
freshly prepared PEC were used in the experiments described. FACS
analysis of isolated PEC showed a >90% staining for the macrophage
surface marker Mac-1. Abs to gp96 (anti-grp94, SPA-850) and to
Hsp70 (SPA-820) were obtained from Stressgen Biotechnologies (Victoria,
Canada). Quantification of TNF- and IFN- was performed as
described previously 13 . For IFN- ELISAs, we used XMG 1.2 (0.5
µg/ml) and RA-6A2 (2 µg/ml) mAbs, and for TNF--specific ELISAs,
we used the Ab pairs G281-2626 (2 µg/ml) and MP6-XT3 (1 µg/ml)
(PharMingen, Hamburg, Germany).
Purification of Hsp70 and gp96
gp96 was purified as described with minor modifications 14 .
Briefly, P13.1 and E.G7 cells were grown in roller bottles to generate
a 20-ml cell pellet. Mouse liver-derived gp96 was purified from 60
g and Hsp70 from 20-g cell pellets. The pellets were homogenized in
four volumes of hypotonic buffer (10 mM NaHCO3 and 0.5 mM
PMSF (pH 7.0)) and centrifuged at 100,000 x g for 90
min. For gp96 purification the 50–70% ammoniumsulfate precipitate was
solubilized in Con A binding buffer (125 mM NaCl, 20 mM Tris-HCl, 1 mM
MgCl2, and 1 mM CaCl2 (pH 7.2)) and was
applied to a Con A/Sepharose column followed by extensive washing. Con
A-bound material was eluted with 10% -methyl-mannoside. The eluate
buffer was exchanged to DEAE-binding buffer (5 mM sodium phosphate and
300 mM NaCl (pH 7.0)), employing PD10 gel filtration units (Pharmacia,
Freiburg, Germany). This partially purified gp96 material was applied
to a DEAE-Sepharose column (Pharmacia) and washed and bound material
was eluted with DEAE-binding buffer containing 700 mM NaCl.
Hsp70 from P13.1, E.G7, and liver cell pellets was purified from the 100,000 x g supernatant by first exchanging the lysis buffer to ADP-binding buffer (20 mM Tris-Acetate, 2 mM MgCl2, 15 mM 2-ME, and 20 mM NaCl (pH 7.0)). The material was applied to an ADP-agarose column (Sigma). The column was washed subsequentlywith ADP-binding buffer containing 500 mM NaCl and 20 mM NaCl. The ADP-bound material was eluted with ADP-binding buffer containing 3 mM ADP. The elution buffer was exchanged to Hsp70-DEAE-binding buffer (20 mM sodium phosphate and 20 mM NaCl (pH 7.0)) again using PD10 columns. The eluate was applied to a DEAE-column and washed. Hsp70 coeluted at a concentration of 150 mM NaCl in DEAE-binding buffer. Fractions of purified gp96 and Hsp70 material were tested in SDS-PAGE and Western blot analysis using mAb specific for Hsp70 and gp96. Fractions containing Hsp70 and gp96 as the major proteins were used for additional experiments. Protein concentration was determined employing Coomassi Plus Protein Reagent (Pierce, Rockford, IL).
Immunization of mice and generation of CTL
Mice were immunized i.p. with 30 µg of gp96 purified from liver, P13.1 cells, or E.G7 cells, 2 x 107 irradiated P13.1 or E.G7 cells in 500 µl PBS, or with PBS only. Mice were sacrificed on day 10. For Hsp70 immunization, mice were injected twice weekly s.c. with 15 µg of Hsp70, 2 x 107 irradiated P13.1, or E.G7 cells in 100 µl PBS, and the mice were sacrificed 7 days after the second immunization. For bulk culture, splenocytes (5 x 106/ml) were restimulated with 1 µM of OVA or ß-gal peptide in upright 10-ml culture flasks. CTL activity was tested in a standard chromium release assay five days after restimulation. For frequency analysis, splenocytes (106/ml) were cultured in 96-well round-bottom culture dishes with 1 µM of ß-gal peptide or irradiated E.G7 cells (5 x 105/ml). Fifty splenocyte lines were tested for Ag-specific lysis on day 6 after restimulation. E.G7- and P13.1-specific lysis was calculated by subtracting the specific lysis of Ag-negative target cells (EL4, P815) from the specific lysis of the Ag-expressing target cell (E.G7, P13.1) for each point.
Stimulation and HSP/peptide representation assays with long-term cultured CTL clones
Indicated types of APC or freshly prepared PEC
(104/well), CTL clones (105/well) and indicated
amounts of HSP, LPS, or peptide were cocultured in 96-well round-bottom
culture dishes for 30 h. A total of 50 µl of the supernatant was
tested for IFN- and TNF- in a standard ELISA. In trans-well
experiments, 0.2-µm anopore membrane transwell strips (Nunc,
Naperville, IL) were employed to block direct cell contact
between CTL and APC within one well.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN- in CTL clones with Ag-negative HSP
preparationsHSP preparations are able to immunize against tumors and to induce CTLs in vivo as described by coworkers of Srivastava 13 and other groups. Moreover, HSP are highly efficient in chaperoning associated peptides into the MHC-I related Ag presentation pathway, as shown in in vitro re-presentation assays 3 . Because in some of our T cell assays (M.B. and A.v.B. unpublished data) the addition of Ag-negative HSP preparations resulted in the induction of lymphokines and proliferation of T cells, we started to analyze a possible T cell-activating potential of HSP that functions irrespective of the HSP-associated peptides.
To this end, we purified Hsp70 and gp96 molecules from the
OVA-transfected cell line E.G7 and control tissues (Fig. 1
). We have recently shown 15 that
Hsp70 and gp96 molecules purified from E.G7 cells are associated with
the major H2-Kb binding epitope OVA257–264. As
shown in Fig. 2, the
H2-Kb/OVA257–264-specific CTL clone 4G3
releases substantial amounts of TNF- when E.G7-purified Hsp70 (Fig. 2A) or gp96 (Fig. 2B) molecules were added to the
cultures. However, the same amount of TNF- could be detected in
cultures when Ag-negative HSP, e.g., liver or RMA-derived Hsp70/gp96,
was titrated into the cell culture system. To exclude the possibility
that the response to Ag-negative HSPs is a characteristic of the 4G3
clone, we repeated the experiment with a ß-gal-specific CTL clone,
0805B. Again, we observed no Ag-specific induction of IFN- after the
addition of P13.1-derived gp96 (Fig. 3,
A and B) and Hsp70 (Fig. 3C), because
Hsp70 and gp96 preparations purified from liver tissue showed a
comparable effect. Although gp96 was purified by Con A-containing
columns, a contamination with Con A can be largely ruled out, because
the stimulating activity in our gp96 preparations could only be
observed in DEAE fractions in which gp96 was present. Moreover,
recombinant His-tagged gp96 purified in the absence of Con A showed
comparable results (S. Moré, M. B., and A.v.B., unpublished
data).
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when compared with
liver-derived gp96 (Fig. 3
B), the inclusion and effective
stimulation of the OVA257–264-specific CTL clone 4G3 in
the same experiment revealed that this induction was independent of
gp96-associated peptides and reflected a higher stimulating
capacity of that particular gp96 preparation. Interestingly, in this
set of experiments we used H-2d-positive macrophages
induced in BALB/c mice as accessory cells. Because the 4G3 clone is
H-2Kb restricted, the induction of IFN- was triggered by
the "wrong" MHC-I haplotype. This result is not due to an
alloreactive reaction of 4G3, because coculture of
H-2d-positive PEC and 4G3 in the absence of HSP did not
induce IFN- or TNF-. Interestingly, Hsp70 purified from liver
stimulated the CTL clones to release IFN- when either ATP-agarose or
ADP-agarose was applied for purification (Fig. 3C). Because
ATP-agarose in contrast to ADP-agarose was described 13 to release
endogenous Hsp70-associated peptides, these results indicate that Hsp70
molecules, irrespective of the complexed peptide-ligands, stimulate 4G3
and 0805B cells. However, the described experimental setup does not
reveal the cytokine source, either T cells or macrophages being
responsible for the secretion of TNF- or IFN-, respectively.
However, we think that the CTL clones in our system are the
cytokine-producing cells, because experiments in which the CTL clones,
following an incubation of 2 h with gp96, were separated from the
adherent macrophages, resulted in TNF- and IFN- release only in
the separated T cell fraction and not in the adherent macrophages (data
not shown). Thus, a short HSP-mediated stimulus is sufficient to induce
cytokine secretion in 4G3 and 0805B T cells.
Another typical read-out system to determine the specific activation of
CTL clones is the chromium release assay, which in contrast to the
quantification of cytokines, lasts only 4 h. Therefore, we
repeated the experiments shown in Fig. 3 with
51Cr-labeled macrophages. As shown in Fig. 4, incubation of gp96 derived from P13.1
and liver cells together with CTL clones resulted in killing
51Cr-labeled macrophages. Again, the CTL-mediated lysis was
not haplotype-restricted, e.g., 0805B cells
(ß-gal/H2-Ld-restricted) lysed to a significant degree
C57Bl/6 macrophages (H-2b).
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), some Hsp70
preparations contained an additional protein band corresponding to 40
kDa. The identity of this protein is unclear, however, Hsp70 is known
to interact with different proteins such as Hsp40 or Hip 16 ,
respectively, with both proteins displaying a molecular mass of 
40
kDa in SDS-PAGE. Fig. 5 shows that the
induction of IFN- in 4G3 cells (Fig. 5C) correlates with
the elution profile of Hsp70 molecules visualized in silver staining
(Fig. 5A) and Western blot analysis (Fig. 5B) and
not, for example, with the copurifying protein (Fig. 5A,
lane 2) that showed a different migration shift (smaller
than Hsp70) and eluted before Hsp70 molecules from the DEAE
column (Fig. 5C, lane 2).
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Although strictly dependent on the presence of accessory cells
(PEC), our previous results suggested that the stimulation of OVA and
ß-gal-specific CTL clones with Hsp70 and gp96 molecules was not MHC
restricted. To confirm this finding, we repeated the experiments with a
broadened set of accessory cells. We incubated Hsp70 and gp96 molecules
together with 4G3 (Fig. 6A) or
0805B (Fig. 6B) cells, respectively, and MHC-I matched and
mismatched macrophages. As shown in Fig. 4, HSP-mediated TNF-
secretion does not depend on the expression of the "correct"
TCR-restricting MHC-I molecules on the accessory cells.
H-2d and H-2b molecules induced TNF- equally
well after conincubation with gp96 and Hsp70 molecules. However, this
HSP-mediated TNF- release was dependent on the type of APC. MHC-I
expressing tumor cell lines (RMA) as well as B cell lines (A20) or a
long-term cultivated macrophage cell line (J774, data not shown) were
not able to induce TNF-. Thus, stimulation of CTL clones with HSPs
obviously requires certain properties of macrophages that are not
present on long-term cell lines but on freshly ex vivo cultured
macrophages.
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induction is not induced by LPS
The release of TNF- and IFN- induced by both HSP
preparations, Hsp70 and gp96, is not dependent on the source of the
purified HSP preparation, e.g., TNF- and IFN- production could be
measured after incubation with both Ag-positive and Ag-negative HSP
preparations. To exclude the possibility that the observed TNF- in
cultures containing macrophages is due to contaminations with
endotoxins in the process of HSP purification, we compared our HSP
preparations to LPS at concentrations that induced comparable TNF-
secretion. We performed two series of additional experiments. In the
first set, we boiled LPS and the HSP preparations prior to the
incubation with CTL clones and macrophages (data not shown). In the
second set of experiments, we incubated CTL clones and macrophages
together and thus allowed cell contact, whereas in a parallel
experiment we prevented direct cell contact with the help of trans-well
stripes (Fig. 7). Either boiling or the
prevention of direct cell contact completely abolished the TNF-
secretion by coincubation with HSPs, whereas the TNF- inducing
effect of LPS in both cases is only mildly (by boiling) or not at all
(in the trans-wells) affected. These experiments largely rule out: 1)
that contaminating endotoxins and 2) that a cell contact-independent
cytokine cross-talk are responsible for the cytokine inducing effects.
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Next, we analyzed the Ag-independent stimulating activity of Hsp70
molecules also in vivo. Two batches of Hsp70 were prepared; one was
derived from E.G7 cells (OVA+) and the second was derived
from normal mouse liver tissue. In a first set of experiments, we
immunized four groups of C57BL/6 mice: the first group were control
mice immunized with PBS (negative control group), the second group
received 2 x 107 irradiated E.G7 cells
(OVA+, positive control group), the third group was
immunized i.p. with E.G7-derived Hsp70, and the fourth group with
liver-derived Hsp70. After 7 days, mice were sacrificed, and
spleen cells were cultured in the presence of antigenic OVA peptide as
described in Materials and Methods. After 6 days of culture,
induced OVA257–264-specific CTLs were tested in in vitro
cytotoxic chromium release assays. As shown in Fig. 8, immunization with E.G7 cells resulted
in a significantly higher frequency of OVA-specific CTL when compared
with PBS. However, priming of OVA-specific CTL with Hsp70 worked
efficiently with both employed Hsp70 preparations; that is, also the
OVA-Ag-negative liver-derived Hsp70 was able to induce OVA-specific
CTL that were able to lyse E.G7 cells (Fig. 8) and to secrete TNF-
and IFN- after coincubation with E.G7 cells (data not shown). The
induced CTL clones were
OVA257–264/H2-Kb-specific, because killing
could be blocked by preincubation with H2-Kb- (but not with
H2-Db) specific mAb (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, HSP-associated peptides seem to reflect the whole or
at least a great part of the interior world of a given cell type,
enabling the induction of a broad spectrum of Ag-specific CTL clones.
Members of the Hsp70 family bind preferentially linear polypeptide
fragments, and therefore represent central components of the protein
folding machinery in the cell 20, 21 . In this context, it could be
shown that gp96, an endoplasmic reticulum resident HSP, is able
to induce crosspriming after immunization with gp96 preparations 22 .
One could imagine that even Hsp70 preparations derived from
OVA-negative tissue are associated with cross-reactive
self-peptides or other ligands that lead to activation of OVA-specific
T cells. In our in vitro experiments, we can exclude the
possibility that cross-reactive "non-OVA" peptides are responsible
for the observed stimulation of the
H-2Kb/OVA257–264-specific CTL clone 4G3,
because stripping of liver gp96 and Hsp70-associated peptides did not
sensitize this clone on RMA target cells (Fig. 3C and Refs.
13 and 15).
Immunization with irradiated cells worked very efficiently in our in
vivo experiments. A tempting working hypothesis would be that these
irradiated APCs do not only display antigenic peptides in context of
MHC-I molecules on their cell surfaces but also release immunogenic
HSP/peptide complexes after cellular breakdown. This release of HSP
molecules could reflect a danger signal 23 , attracting many different
T cells and other members of the immune system to start a broad primary
immune response. However, replacing ADP-purified Hsp70 preparations
(e.g., still complexed with endogenous peptides) with ATP-purified HSP
molecules (e.g, peptide-negative) using a method first described by
Tamura et al. 13 resulted in comparable results in vivo and in
vitro arguing against a central role for peptides at least for the
OVA-system (data not shown and Fig. 3C).
Most of the experiments published so far that concentrated on the induction of CTL or the protection from tumors employed gp96 preparations. However, recent reports also started to analyze the function of in vitro reconstituted Hsp70/peptide complexes 24 or used Hsp70 fusion proteins 25 . Interestingly, in vitro complexation of Hsp70 molecules with OVA-peptides led to the induction of OVA-specific CTLs. Because E.G7-purified Hsp70 (despite being associated with OVA257–264 15) did not specifically generate an OVA-specific CTL response, one could argue that in vitro reconstituted Hsp70, due to the high number of associated synthetic peptides, has a higher immunizing capacity. Our in vivo data generally show that Hsp70 has a broader, and at least for OVA, unspecific stimulating capacity, whereas gp96 administration induced a specific amount of Ag-specific CTLs (in case of ß-gal) or displayed no induction at all (in the case of E.G7-derived gp96). It may be worthwhile to see whether differences in the immunization capacities of Hsp70 and gp96 molecules can be further proven, which may help us to better understand the powerful immunological potential of HSP.
The mechanism that enables Hsp70 and gp96 molecules to trigger T cells
in vitro is not understood. Because minimal amounts of HSPs are
suffcient to induce protection in tumor-challenging protocols, putative
Hsp70 and gp96 receptors on the cell surface that introduce the uptaken
HSP/peptide complexes directly into the MHC-I pathway have been
postulated. However, so far there is no evidence for the existence of
such receptors. Obviously the choice of the correct accessory cells is
very important for detecting HSP-induced re-presentation of HSP/peptide
complexes. To this end all long-term cultured cell lines used as
accessory cells did not work in our assays; only freshly isolated
macrophages responded to the coincubation with HSP molecules. Although
pristane-induced PEC contain a few contaminating T cells (<5%), these
T cells do not seem to be responsible for the observed effects, because
cultures of PEC and HSP without additional T cell clones did not
contain significant amounts of TNF- and IFN-. It may well be that
only these ex vivo purified macrophages express surface structures that
are able to interact with HSP complexes, whereas in vitro cultured
cells have lost such expression markers. The induction of TNF- and
IFN- is not dependent on the expressed MHC-I haplotype. Ongoing
studies are concentrating on the identification of molecules that
mediate or at least enhance the HSP-induced stimulation of T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arne von Bonin, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nochtstr. 74, D-20359 Hamburg, Germany. E-mail address:
3 Abbreviations used in this paper: HSP, heat shock protein; ß-gal, ß-galactosidase; VSV, vesicular stomatitis virus; PEC, peritoneal exudate cell(s).
Received for publication July 6, 1998. Accepted for publication December 7, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. Feng, Y. Zeng, L. Whitesell, and E. Katsanis Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity Blood, June 1, 2001; 97(11): 3505 - 3512. [Abstract] [Full Text] [PDF]
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D. Skokos, S. Le Panse, I. Villa, J.-C. Rousselle, R. Peronet, B. David, A. Namane, and S. Mecheri Mast Cell-Dependent B and T Lymphocyte Activation Is Mediated by the Secretion of Immunologically Active Exosomes J. Immunol., January 15, 2001; 166(2): 868 - 876. [Abstract] [Full Text] [PDF]
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C. Castelli, A.-M. T. Ciupitu, F. Rini, L. Rivoltini, A. Mazzocchi, R. Kiessling, and G. Parmiani Human Heat Shock Protein 70 Peptide Complexes Specifically Activate Antimelanoma T Cells Cancer Res., January 1, 2001; 61(1): 222 - 227. [Abstract] [Full Text]
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C. Maranon, L. Planelles, C. Alonso, and M. C. Lopez HSP70 from Trypanosoma cruzi is endowed with specific cell proliferation potential leading to apoptosis Int. Immunol., December 1, 2000; 12(12): 1685 - 1693. [Abstract] [Full Text] [PDF]
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 D. M. Hall, L. Xu, V. J. Drake, L. W. Oberley, T. D. Oberley, P. L. Moseley, and K. C. Kregel Aging reduces adaptive capacity and stress protein expression in the liver after heat stress J Appl Physiol, August 1, 2000; 89(2): 749 - 759. [Abstract] [Full Text] [PDF]
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C.-H. Chen, T.-L. Wang, C.-F. Hung, Y. Yang, R. A. Young, D. M. Pardoll, and T-C. Wu Enhancement of DNA Vaccine Potency by Linkage of Antigen Gene to an HSP70 Gene Cancer Res., February 1, 2000; 60(4): 1035 - 1042. [Abstract] [Full Text]
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Q. Huang, J. F.L. Richmond, K. Suzue, H. N. Eisen, and R. A. Young In Vivo Cytotoxic T Lymphocyte Elicitation by Mycobacterial Heat Shock Protein 70 Fusion Proteins Maps to a Discrete Domain and Is Cd4+ T Cell Independent J. Exp. Med., January 17, 2000; 191(2): 403 - 408. [Abstract] [Full Text] [PDF]
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 C. Thery, A. Regnault, J. Garin, J. Wolfers, L. Zitvogel, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena Molecular Characterization of Dendritic Cell-Derived Exosomes: Selective Accumulation of the Heat Shock Protein Hsc73 J. Cell Biol., November 1, 1999; 147(3): 599 - 610. [Abstract] [Full Text] [PDF]
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J. Robert, A. Menoret, and N. Cohen Cell Surface Expression of the Endoplasmic Reticular Heat Shock Protein gp96 Is Phylogenetically Conserved J. Immunol., October 15, 1999; 163(8): 4133 - 4139. [Abstract] [Full Text] [PDF]
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), pristane induced H-2b-positive
macrophages (2.5 x 105/ml, 
), or APC and 4G3 cells
together (
). Shown is a typical result of four independent
experiments.
) for 24 h with titrated amounts
of gp96 purified from liver (A) or P13.1 cells
(B). Shown is the induced IFN-
)
incubated without CTL clones in a 4-h standard chromium release assay.
Specific lysis (triplicate values) is shown on the
y-axis. Lysis of unmodified PEC was <15% and is
subtracted from each value. Comparable results were obtained with
H2d-positive PEC (data not shown).
