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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pediatrics, Steele Memorial Children’s Research Center, University of Arizona, Tucson, Arizona 85724-5073
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A variety of tumor types growing as s.c. masses have been reported to be responsive to tumor-derived chaperone vaccination (1) . However, none of these studies have addressed the effectiveness of chaperone vaccines in generating antitumor immunity against disseminated hematological malignancies such as leukemias or lymphomas. This report describes our attempts to elicit antitumor immune responses via chaperones purified from the spontaneously derived murine A20 B-cell leukemia/lymphoma (11) . When A20 cells are injected i.v. into BALB/c mice, they induce a disseminated disease characterized by infiltration of lymph nodes, liver, and spleen and the presence of malignant cells in bone marrow and peripheral blood. This leukemia is radioresistant, with even myeloablative doses of irradiation followed by syngeneic bone marrow transplantation failing to cure A20 bearing mice (data not shown; Ref. 12 ). A20 is poorly immunogenic; vaccination with irradiated wild-type tumor cells confers little protection to subsequent tumor challenge. Systemic T-cell immunity against A20 may be generated, however, following immunization with genetically modified tumor cells engineered to secrete GM-CSF and other cytokines (13) .
Herein we report a method for sequential purification of the major immunologically active chaperone proteins HSP70, HSP90, grp94/gp96, and calreticulin from a single A20 tumor sample and demonstrate the relative efficacy of these protein preparations in generating specific antitumor responses in this aggressive murine leukemia model. Syngeneic GM-CSF-secreting fibroblasts were injected at the immunization site in an attempt to stimulate antigen uptake and presentation by professional APCs. Interestingly, production of GM-CSF at the vaccination site by transduced 3T3 fibroblasts actually abrogated the protective effect.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Cell Lines.
A20 is a B-cell leukemia/lymphoma that arose spontaneously in an old
(>15 months) BALB/c mouse (11)
. BDL-2 is a murine B-cell
lymphoma line (H-2d) established by the long-term
in vitro culture of murine lymphoid tissue and was kindly
provided by Dr. Jonathan Braun, University of California, Los Angeles
School of Medicine (Los Angeles, CA; Ref. 14
). The 3T3
fibroblasts were purchased from American Type Culture Collection
(Manassas, VA). All tissue culture reagents were from Life
Technologies, Inc. (Gaithersburg, MD). The cell lines were cultured at
37°C under 5% CO2 in air using RPMI media
containing 10% heat-inactivated FCS and supplemented with 2
mM glutamine, 100 units/ml penicillin, 100
µg/ml streptomycin sulfate, 0.025 µg/ml amphotericin B, 0.5x MEM
nonessential amino acids, 1 mM sodium pyruvate,
and 50 µM 2-mercaptoethanol.
Transduction of 3T3 Cells.
GM-CSF gene transfer was performed using the MFG-GM-CSF
retroviral vector (kindly provided by Drew Pardoll, Johns Hopkins
University, Baltimore, MD), which is a Moloney murine leukemia
virus-based vector that does not contain selection genes (15
, 16)
. Supernatants from the retroviral producer cell line, CRIP,
were collected and used to transduce 3T3 cells in the presence of 8
µg/ml polybrene for 4 h and repeated after 24 h.
GM-CSF Bioassay.
The GM-CSF-dependent cell line FDCP2–1D was used to determine the
amount of GM-CSF bioactivity present in the supernatants of transduce
3T3 cells (17)
. Samples were added in triplicate to
96-well flat-bottomed plates (Costar, Cambridge, MA) and serially
diluted 3-fold. FDCP2–1D cells (5000 cells/well), washed free of
GM-CSF, were then added to the plates. The microtiter plates were
incubated for 24 h at 37°C and 5% CO2,
then pulsed for 24 h with 2 µCi
[3H]thymidine (Dupont New England Nuclear,
Boston, MA). The cells were then harvested using a Packard plate
harvester, and the radioactivity was measured on a Packard ß counter.
The GM-CSF concentrations were calculated by comparing to a known
GM-CSF standard (PeproTech, Inc., Rocky Hill, NJ).
Purification of Tumor-derived Chaperone Proteins.
A20 cells were prepared for injection by washing and resuspending them
in HBSS. Tumors >1 cm in diameter were surgically harvested after
euthanizing the mice. Resected tumor tissue was homogenized in a
hypotonic buffer plus protease inhibitors. Fig. 1
schematically outlines the purification
and may prove helpful in following the steps described from this point.
Following a low-speed centrifugation (12,000 x g; 30
min; 4°C) to remove debris, a high-speed supernatant (100,000 x
g; 90 min; 4°C) was obtained. Albumins were removed via a
Cibacron Blue 3GA (Sigma, St. Louis, MO) resin. The Ciba Blue
flow-through material was chromatographed over ConA-Sepharose (Sigma,
St. Louis, MO); the lectin column was eluted using 10%
-D-methylmannose hypotonic buffer. Elution
fractions containing grp94/gp96 (as determined by SDS-PAGE and Western
blotting) were pooled and dialyzed into a Tris-acetate/NaCl buffer.
Grp94/gp96 was further purified on a Hi Trap Q strong anion exchange
column (Pharmacia Biotech, Piscataway, NJ), which was eluted in NaCl.
Fractions containing grp94/gp96 were finally purified by Hi Trap
Heparin chromatography (Pharmacia Biotech) treated as an anion exchange
resin and eluted in an NaCl gradient. Fractions containing the purified
gp96 were identified by SDS-PAGE and Western blotting. Flow-through and
wash fractions from the ConA column were pooled and differentially
precipitated with ammonium sulfate; precipitated proteins were pelleted
by centrifugation, resuspended in Tris-Acetate/NaCl, and dialyzed.
Proteins from the 70% ammonium sulfate cut were separated on a
DEAE-Sephacel resin (Pharmacia Biotech). Fractionated proteins were
analyzed by SDS-PAGE and Western blotting. Fractions enriched for
proteins of interest were pooled and concentrated. HSP70 was purified
from DEAE-pooled fractions via ADP-agarose affinity chromatography (N-6
linkage, Sigma, St. Louis, MO) essentially as described previously
(18)
. HSP70 was identified and purity was assessed by
SDS-PAGE and Western blotting. HSP90 was further purified from DEAE
elution fractions by hydroxyapatite chromatography. Proteins were
dialyzed into phosphate buffer, concentrated, loaded onto the column,
and eluted in a step gradient of 100 mM, 200
mM, 300 mM, and 400
mM sodium phosphate (pH 7.0). Fractions
containing HSP90 were identified via Western blotting; positive
fractions were pooled, and the buffer exchanged to Tris-acetate/NaCl.
The pooled material was chromatographed on a Hi Trap Q column and
eluted in NaCl. Fractions containing purified HSP90 were identified by
SDS-PAGE and Western blotting. Calreticulin was purified from the 85%
ammonium sulfate precipitate and high salt DEAE elutions by dialysis
into 10 mM
3-[N-morpholino]propanesulfonic acid, 100
mM NaCl (pH 7.1), and precipitation with 10
mM ZnCl2. The precipitate
was recovered by centrifugation, resuspended and dialyzed into
Tris-acetate/NaCl, and chromatographed on Hi Trap Q, eluted in NaCl.
Fractions containing purified calreticulin were identified by
SDS-PAGE and Western blotting.
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In Vivo Immunoprotection Experiments.
BALB/c mice were immunized s.c. in the flank with A20-derived HSP70,
grp94/gp96, calreticulin, or HSP90 in 0.2 ml PBS. Control mice received
PBS only. Mice were vaccinated twice at weekly intervals
(i.e., day -14 and day -7). Some experiments involved
prevaccination using 3T3 cells transduced (or not) with the gene
encoding murine GM-CSF. Fibroblasts were then injected s.c. on day -15
and day -8, whereas chaperone vaccinations were given at the same site
on day -14 and day -7. Seven days after the last immunization (day
0), mice were challenged via tail vein injection with 1 x
106 viable A20 or BDL-2 B-cell leukemia/lymphoma
cells using a 27-gauge needle. Both A20 and BDL-2 cells used for
challenge were harvested from tumor-bearing mice 2–3 h before
injection. s.c. tumors were removed under sterile conditions, placed in
PBS, finely minced with a scalpel, gently crushed with the piston of a
syringe, gathered in a pipette, filtered through a 100-µm Nytex mesh,
washed twice in PBS, and resuspended at a concentration of 5 x
106/ml.
Statistical Analysis.
The Kaplan-Meier product-limit method was used to plot the survival of
mice inoculated with the B-cell leukemias and the log-rank statistic to
test differences between groups (21
, 22)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The protein purification scheme outlined herein represents a
modification of previous techniques used to purify chaperones. These
methods have been revised and refined to allow stepwise progression
through the protocol with high efficiency and recovery. Additionally,
the procedure employs relatively inexpensive separations, such as
centrifugation and precipitation, as well as "low tech"
conventional and affinity chromatographies. Fig. 1A
outlines
the purification scheme, and Fig. 1B
shows SDS-PAGE analysis
(left) and Western blots (right) of the resulting
final materials. Each of the purified proteins was subjected to Western
blotting with antibodies against each of the other chaperones, and no
cross-contamination was detected (data not shown).
Concerns about the use of ConA chromatography have arisen previously
(23)
. The assertion is that low levels of ConA
contamination insidiously present in the final purified components may
be the actual source of a generalized immune response. We do not
believe that such a scenario is true for several reasons:
(a) The chaperone proteins provide specific immunity only
against the tumor that served as the chaperone source (see Table 1
) and not against a different syngeneic
B-cell malignancy (BDL-2); (b) We have purified and
identified by NH2-terminal amino acid sequencing
a protein that copurifies with calreticulin until separation in the
final step (including passage through a ConA column). This protein,
identified as mouse serum albumin, is ineffective as a vaccine against
A20 tumor challenge (data not shown); (c) We have also
purified A20 HSP70 to a high degree using only centrifugation,
ADP-agarose affinity chromatography, and anion exchange chromatography,
thus bypassing the ConA column. In survival experiments, these
preparations provide protection to mice from tumor challenge that is
indistinguishable from A20 HSP70 purified by the longer protocol
(i.e., including the lectin chromatographic step). These
observations lead us to conclude that ConA immune stimulation is not a
factor in our assays.
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To determine whether or not the tumor-derived chaperone proteins purified by this protocol were effective in stimulating immune responses against A20 leukemia, syngeneic BALB/c mice were immunized with purified chaperone preparations (HSP70, HSP90, grp94/gp96, and calreticulin) from an A20 tumor source on days -14 and -7, followed by i.v. challenge with A20 on day 0.
Fig. 2A
depicts Kaplan-Meier
plots comparing the protective effect of different chaperone
vaccinations. In these experiments, mice were given two 20-µg doses
of purified chaperones before tumor injection. HSP70, grp94/gp96, and
calreticulin all provided significant improvement in survival over
controls, with A20-derived HSP70 being the most effective chaperone
protein followed by grp94/gp96 and calreticulin. A20-derived HSP90,
given in 30-µg doses, also provided substantial protection compared
to PBS-treated controls (Fig. 2B
).
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. Naïve mice
were i.v. challenged with 106,
105, or 104 A20 cells. Of
note, A20 cells derived from in vivo-propagated tumor cells
are more tumorigenic than those cultured in vitro (data not
shown; Ref. 30
). Only in vivo grown B-cell
leukemia/lymphoma cells were use in our studies. Based on the survival
curves generated, A20-derived HSP70 vaccinations provide protection in
terms of increased survival equivalent to at least a 2-log reduction in
tumor burden (Fig. 3)
. There is obvious clinical significance in the
degree of immune-mediated cytotoxicity induced by chaperone
protein vaccination. If used in the setting of minimal residual
disease, such as following autologous stem cell transplantation,
chaperone protein vaccination may increase leukemia/lymphoma cure rates
without toxicity.
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). However, on day 0, the vaccinated mice and untreated
controls were i.v. challenged with the BDL-2 B-cell leukemia/lymphoma,
which is syngeneic to BALB/c mice but antigenically unrelated to A20.
As outlined in Table 1
, A20-derived chaperone protein vaccinations did
not generate significant protection against BDL-2. These data confirm
that the immunity provided by A20-derived chaperone proteins is
tumor-specific.
Localized GM-CSF Cytokine Production Inhibits Chaperone-induced
Protection.
Because tumor-derived chaperone protein vaccinations are capable of
generating CTL responses against the tumor of origin, it is posited
that professional APCs are involved in transiting antigenic peptides
from the vaccinating chaperones into effective antigen presentation
pathways. We sought to augment the immune response resulting from
chaperone injections by providing a localized production of GM-CSF,
which is known to stimulate dendritic cells and macrophages at the site
of immunization (15
, 31)
. 3T3 fibroblasts were transduced
with the murine GM-CSF gene and were found to produce 3–5 ng/ml of the
cytokine/106 cells/24 h (data not shown). On days
-15 and -8, GM-CSF-transduced 3T3 fibroblasts or unmodified 3T3 cells
were injected s.c. into a marked shaven site of each mouse. Twenty four
h later (days -14 and -7), mice were immunized with chaperone
preparations (20 µg of either HSP70 or grp94/gp96) into the same
site. i.v. A20 tumor challenge was given on day 0, and survival was
monitored. Surprisingly, production of GM-CSF at the vaccination site
abrogated the effects of chaperone immunization (Fig. 4, A
and B).
Similar inhibitory effects of GM-CSF were evident in the case of
calreticulin vaccination (data not shown). As discussed later, this may
be secondary to unopposed production of GM-CSF inhibiting T-cell
responses by dysregulating APC maturation (32)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the immunological activities of tumor-derived HSP70, grp94/gp96, and HSP90 family members have been known for some time, calreticulin is a newcomer to this field. Basu et al. (23) recently demonstrated that calreticulin purified from a methylcholanthrene-induced fibrosarcoma when used as a vaccine confers protection to subsequent s.c. tumor challenge. Moreover, Nair et al. (25) reported that dendritic cells pulsed with calreticulin isolated from B16 melanoma or ovalbumin-transfected EL-4 thymoma elicits tumor-specific CTL responses as assessed by in vitro cytotoxicity assays. These investigators ranked calreticulin as equivalent or even better than grp94/gp96 in the assays used. In contrast, in our hands, calreticulin was not as effective against A20 leukemia when compared to HSP70 and gp96/grp94. The number of subcellular localizations and proposed roles for calreticulin within (and outside) the cell have risen dramatically over the past few years (33, 34, 35, 36, 37) . The discovery of calreticulin’s involvement in antigen processing is quite recent, however (38, 39, 40) . It is possible that calreticulin may be involved in this mechanism of peptide retention, and as such, it is a reasonable candidate for a chaperone of potentially antigenic peptides from a tumor source.
The molecular mechanisms of antigen capture, antigen presentation, and effector cell stimulation following exogenous administration of tumor-derived chaperone proteins are not clear. Suto and Srivastava (9) demonstrated the need for APCs in chaperone-based immune responses in tumor rejection, and in vitro studies using chaperone-pulsed APCs further elucidated the role of APCs (3 , 25) . Actual uptake of an exogenously delivered chaperone protein by APCs was observed recently (2) . Together, theses studies imply the presence of receptors for chaperones, at least on the surfaces of APCs. Competition experiments indicate that the chaperone proteins HSP70 and grp94/gp96 may have different receptors (2) .
In view of the importance of professional APCs in generating tumor-derived chaperone-based immune responses, we introduced syngeneic GM-CSF-producing fibroblasts at the vaccination site in an attempt to augment the immune response by stimulation and local and expansion of APCs before injection of chaperone protein preparations. Surprisingly, the protective effect of tumor-derived chaperone vaccinations was abrogated under these conditions. Bronte et al. (32) have reported inhibition of CD8+ T-cell responses in tumor microenvironments where the tumor secretes GM-CSF. The inhibition appears to occur due to failure of certain APC progenitors to fully mature in the presence of GM-CSF alone. An inhibitory population of cells is present (CD11b+/Gr-1+) causing an impairment of CD8+ T-cell function and even inducing apoptotic T-cell death (32) . This inhibition could be overcome with the administration of IL-4, indicating that a balance between the two cytokines is necessary to fully mature the APCs. These mechanisms certainly seem plausible in our experimental setting.
The concept of using chaperone proteins purified from tumors as vaccines against those tumors suggests that a multivalent response against the full repertoire of potentially antigenic peptides in a tumor may be important in generating effective immunoprotection. We suggest that to induce the maximally effective antitumor response, it may be necessary to use several types of chaperones that could be complexed with a wide variety of tumor-derived peptides. Our purification scheme enables one to purify a variety of such chaperones from a single tumor source; thus isolated, one may quantify the effects of each chaperone in terms of generating an antitumor response, and perhaps, one may determine the mechanisms behind that response. Ongoing studies in our laboratory are presently examining the benefits of multiple chaperone protein vaccines from the same tumor used alone or in combination. An understanding of how tumor-derived chaperones may differ in eliciting tumor-specific T-cell responses will undoubtedly prove useful in designing effective combinations of chaperones for anticancer treatments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the American Cancer Society
Grant IM-785, the Arizona Disease Control Research Commission, the
W. M. Keck Foundation, the Enid and Mel Zuckerman Fund, the Arizona
Elks Program in Transplantation Research, and the Michael Landon
Fund. 
2 To whom requests for reprints should be
addressed, at University of Arizona, Department of Pediatrics, 1501
North Campbell Avenue, P. O. Box 245073, Tucson, AZ 85724-5073.
Phone: (520) 626-6527; Fax: (520) 626-4220; E-mail: katsanis{at}peds.arizona.edu
3 The abbreviations used are: APC,
antigen-presenting cell; GM-CSF, granulocyte macrophage
colony-stimulating factor; ConA, concanavalin A; HSP, heat
shock/chaperone protein; ns, not significant.
Received 6/16/99; revised 12/ 7/99; accepted 12/ 8/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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