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*
Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial Cancer Research Fund Oncology Unit, Imperial College of Science and Medicine, Hammersmith Hospital, London, United Kingdom;
Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905;
Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy; and
Department of Experimental Medicine and Pathology, Second Chair of Pathology, University of Rome La Sapienza, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, and
IL-12) and enhances immunogenicity via a T cell-mediated mechanism. In
addition, the protection conferred by hsp70 is both tumor and cell
specific. We also demonstrate that hsp70 targets immature APC to make
them significantly more able to capture Ags. This is likely to optimize
cross-priming of the infiltrating APC with tumor Ags, which are
simultaneously being released by the dying cells. In addition, using an
Myc epitope-tagged hsp70 expression vector, we present evidence that
hsp70 released from dying tumor cells is taken up directly into DCs and
may, therefore, be involved in direct chaperoning of Ags into DCs.
Taken together, our data suggest that hsp70 induction serves to signal
the immune system of the presence of an immunologically relevant
(dangerous) situation against which an immune reaction should be
raised. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using gene transfer of the HSV thymidine kinase (HSVtk) suicide gene to the murine melanoma B16 model, we demonstrated that in vivo killing of this otherwise poorly immunogenic tumor generates a potent antitumor immunity (20, 21). Other groups have shown similar results (22, 23, 24, 25, 26). Furthermore, when B16 cells are killed in vivo by GCV, a highly immunostimulatory intratumoral environment is created, as shown by the expression of Th1-type profile of cytokines and the appearance of a dense infiltrate of T cells and macrophages (27). More recently, we showed that the physiological and biochemical mechanisms by which tumor cells are killed in vivo may directly affect their immunogenicity, such that the more physiologically "dangerous" the method that tumor cells can be killed, the greater the chance that immune reactivity will be raised against them (2, 17, 28).
During our studies to identify how the immune system might perceive
these different mechanisms of cell death as significant or not, we
observed that cells in which killing was less apoptotic (and
correspondingly more immunogenic) showed an increase in the expression
of inducible hsp (2). Thus, hsp induction may directly
influence whether the relevant cells of the immune system, such as DC,
become appropriately activated or not (29). Similarly,
previous reports have shown that hsp may mediate effective antiviral-
or antitumor-specific immunity (19, 30, 31) deriving at
least in part from antigenic peptides chaperoned by the hsp into a
subset of APC in vivo (32, 33, 34, 35, 36). In addition, expression of
hsp in tumor cells may also enhance immunogenicity by direct
presentation of Ags to 
T cells (37, 38) and, by
analogy from gene transfer experiments using highly conserved bacterial
hsp, may also act as a potent immunogenic adjuvant in their own right
(39, 40).
Therefore, our data concerning the suicide gene killing of tumors suggest that tumor immunogenicity might be manipulated therapeutically by ensuring that tumor cells are recognized in vivo under the appropriate immune stimulatory conditions such as necrotic cell death and/or in the context of immune-enhancing danger signals. In this report we have investigated how the expression of the inducible hsp70 signals to the immune system the presence of an immunologically relevant (dangerous) situation against which an immune reaction should be raised.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B16 F1 cell line used in this study has been described previously (41). CMT93 is a murine colorectal tumor line derived from a C57/BL mouse (42). Cell lines were monitored routinely and were found to be free of Mycoplasma infection; they were grown in DMEM supplemented with 10% (v/v) FCS, 4 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin sulfate. For cell killing in vitro, this medium was supplemented with ganciclovir (GCV; CYMEVENE, Roche, Welwyn Garden City, U.K.) to a final concentration of 5 µg/ml.
B16-hsp70 and CMT93-hsp70 cells have been described previously (2). Briefly, murine melanoma B16.F1 cells or murine colorectal carcinoma cells CMT93 were transfected with the cDNA of the inducible hsp70 gene cloned downstream of the CMV promoter in the PCR3 eukaryotic expression vector (Invitrogen, San Diego, CA). Pooled populations of G418r cells were isolated, and populations of transfected cells expressing levels of hsp70 mRNA comparable to those induced by treatment of B16tk cells with GCV in vitro for 48 h were isolated and used for in vivo studies. The hsp70/Myc tag expression vector was constructed by PCR of hsp70 incorporating NheI and BamHI restriction sites. The product was cloned into the pcDNA3.1(-)/Myc-His A vector (Invitrogen), and the correct cloning was confirmed by sequencing. B16 cells were transfected with the hsp70/Myc tag expression vector and selected in G418, and clones were picked. Expression of Myc was confirmed by Western blot. Tumor cells were metabolically labeled by culturing 2 x 106 cells with 9 MBq of [35S]methionine for 16 h. Lysates of these cells were produced by freeze-thawing four times, to give an equivalent of 3 x 105 cells/ml.
DC culture
DC were cultured from the bone marrow of C57BL mice according to
a protocol modified from that described by Inaba et al.
(43). Bone marrow was flushed from femurs and tibias,
resuspended at 1 x 106 cells/ml, and
cultured in 75-cm2 flasks (25 ml/flask) in RPMI
1640 supplemented with 10% FCS, 5 x 10-5
M 2-ME and different concentrations of GM-CSF derived from X63-GMCSF
cell supernatants. X63 cells transfected with the mouse GMCSF gene (D.
Gray, Royal Postgraduate Medical School, London, U.K.) were cultured,
and supernatant was harvested by Central Cell Services, Imperial Cancer
Research Fund (London, U.K.). Culture medium was changed on day 3 of
culture, and on days 7–9, cells in suspension were collected together
with those dislodged by vigorous pipetting from aggregates on the
bottom of the flasks. The yield of DC by this protocol was 
1 x
106/mouse. To test the phagocytic ability of the
DC, 2 x 105 cells were incubated with
4 x 107 2-µm diameter green fluorescent
latex beads (Sigma, Poole, U.K.) for 30 min at 37°C. Cell membranes
were counterstained with red fluorescent membrane stain Dil (Dil
C18(3), Molecular Probes, Eugene,OR) at 2 µg/ml
for 30 min at 37°C, then fixed with 4% formaldehyde. The cells were
visualized using a Zeiss confocal microscope (Zeiss, New York,
NY).
Western blots
Tumor cells or DC were lysed in lysis buffer (50 mM Tris (pH 7.4), 1% Triton X-100, 5 mM EDTA, and 150 mM NaCl), and lysates were heated at 100°C for 2 min with reducing loading buffer. The lysates were run on 10% SDS-PAGE gels at 45 mA for 45 min. The proteins were transferred to nitrocellulose for 1 h at 25 V. Blots were blocked in PBS/5% skimmed milk powder and incubated with 9E10 anti-Myc Ab (Imperial Cancer Research Fund) at 5 µg/ml for 1 h, followed by incubation with rabbit anti-mouse HRP conjugate (Dako, Glostrup, Denmark). Bands were revealed using the enhanced chemiluminescence kit (Amersham, Aylesbury, U.K.), following the manufacturer’s instructions, by exposing the blot to Kodak film (Eastman Kodak, Rochester, NY).
Assay for NK cell activity
Splenocytes were extracted from spleens of naive mice by teasing, and RBCs were lysed with ammonium chloride (0.87%) for 2 min. NK (or lymphokine-activated killer) cells were generated from the splenocytes by culturing 1 x 106 cells/ml with 500 U/ml of recombinant murine IL-2. RPMI medium used was supplemented with 2 mM glutamine, 50 mM 2-ME, and 10% FCS. NK cells were harvested after 5 days.YAC-1 cells (which are highly susceptible to lysis by NK cells), B16, and B16-hsp70 tumor cells were incubated with sodium [51Cr]chromate for 1 h at 37°C, washed, and resuspended at 105 cells/ml. One hundred microliters of labeled cells were added to wells in round-bottomed 96-well plates. NK cells were added in triplicate to the target cells to obtain E:T cell ratios of 100, 50, and 25:1. Wells with targets but no effector cells added indicated the level of spontaneous lysis (S), while wells to which 0.1 mM NaOH was added caused maximal lysis (M). After incubation at 37°C for 4 h, the plate was centrifuged at 600 x g for 5 min, and 100 µl of supernatant was aspirated and counted using a gamma counter (Beckman, Fullerton, CA). The percentage of specific lysis was calculated as: (effectors - S)/(M - S) x 100.
In vivo studies
All procedures were approved by the Imperial Cancer Research Fund animal research committee. C57BL/6 or T cell-deficient nude mice were obtained from colonies bred at the Imperial Cancer Research Fund. Mice were age and sex matched for individual experiments. To establish s.c. tumors, 1–2 x 105 B16 or B16-hsp70 cells or 2 x 106 CMT93 or CMT93-hsp70 cells were injected s.c. (100 µl) into the flank region; these were the minimum doses required to produce 100% tumor take reproducibly in the mice used in this study. Animals were examined daily until the tumor became palpable, whereafter the diameter, in two dimensions, was measured three times weekly using callipers.
Tumors that were established from the primary inoculation were excised
when the size was 1.0 x 1.0 cm. Mice were then rechallenged
with s.c. injection of 1–2 x 105 (B16) or
2 x 106 (CMT93) parental cells on the
opposite flank. Mice that had rejected the initial inoculation of tumor
cells were also rechallenged. All groups of mice in any one individual
experiment were rechallenged on the same occasion using the same
preparation of cells. The animals were rechallenged 14 days following
the most recent surgical excision that had been performed on any mouse
in the cohort. A naive group of mice was also injected with these cells
at the same time. Animals were examined daily until the tumor became
palpable, whereafter the diameter, in two dimensions, was measured
three times weekly using callipers. Animals were killed when tumor size
was 1.0 x 1.0 cm in two perpendicular directions.
Detection of cytokine RNA from tumors using RT-PCR
Tumor samples removed from animals were snap-frozen in liquid nitrogen to ensure conservation of the RNA. RNA was prepared by homogenization of the tumor with RNAzol (Biogenesis, Bournemouth, U.K.) followed by RNA extraction according to the manufacturer’s instructions. RNA concentrations were measured, and 1 µg of total cellular RNA was reverse transcribed in a 20-µl volume using oligo(dT) as a primer and Moloney murine leukemia virus reverse transcriptase (Pharmacia LKB Biotechnology, Milton Keynes, U.K.). A cDNA equivalent of 1 ng of RNA was amplified by the PCR using primers specific for individual murine cytokines. PCR was performed in a 50-µl reaction mixture with 250 µM of each dNTP, 100 nM of primers, 5 µl of 10x buffer (HT Biotechnology, Cambridge, U.K.), and 1 U of super Taq DNA polymerase (HT Biotechnology) using 30 cycles (94°C, 1-min denaturation; 58°C, 1.5-min annealing; and 72°C, 2-min extension). The reaction mix (25-µl samples) were analyzed by agarose gel electrophoresis (1%) in TAE buffer containing 0.2 µg/ml ethidium bromide. In all experiments, a mock PCR (without added DNA) was performed to exclude contamination. To exclude carryover of genomic DNA during the RNA preparation step, controls were also conducted in which the reverse transcriptase enzyme was omitted.
Immunohistochemistry
B16-hsp70 or B16 tumors were allowed to grow s.c. in C57BL mice to 1.0 x 1.0 cm, surgically excised, snap-frozen in liquid nitrogen, and stored at -80°C until sectioning. Five-micron cryostat sections were fixed in acetone and immunostained with rat anti-mouse mAbs against CD45 (clone M1/9.3.4), CD8 (clone 53.6.72), CD4 (clone GK1.5), Mac-3 (clone M37/84.6.34), and MHC-II (clone B21-2) from American Type Culture Collection (Manassas, VA); GR1 (clone RB6-8C5) CD11c (clone HL3) from PharMingen (San Diego, CA); and DEC205 (NDLC-145, provided by Ralph Steinman, Rockefeller University, New York, NY) and with hamster anti-mouse AB: CD3 (clone145-2C11), CD54 (clone3E2), and CD80 (clone 16-10A1) from PharMingen. Sections were preincubated with rabbit or hamster serum and sequentially incubated with optimal dilution of primary Abs, biotinylated rabbit anti-rat or rat anti-hamster (group I or II) IgGs and streptavidin-HPR (PharMingen). Each incubation lasted 30 min and was followed by a 10-min wash in Tris-buffered saline. Sections were then stained with 0.03% H2O2 and 0.06% diaminobenzidine (Sigma) in Tris-buffered saline for 2–3 min, washed in tap water, and finally lightly counterstained with hematoxylin. The number of immunostained cells was determined by light microscopy at x400 magnification in five random fields with the help of a 1-mm2 grid, and is given as cells per square millimeter of tissue (mean ± SD)
Statistics
Data from the animal studies were analyzed by the log-rank test (44).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we demonstrated that the tumorigenicity of B16 cells
engineered to express hsp70 is not significantly altered relative to
that of the parental line when grown in syngeneic C57BL mice. However,
when animals vaccinated with live hsp70-expressing B16 were
rechallenged with parental cells, up to 60% of the animals were
protected over long time periods (>60 days following challenge)
(2) (Table I
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Similar experiments with CMT93 cells demonstrated that hsp70 expression
not only increases the immunogenicity of the parental cell line but
also decreases its tumorigenicity (Table I).
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). As with growth in the immunocompetent
syngeneic mice, B16-hsp70 tumors grew at the same rate as the parental
tumors (Fig. 1A). In contrast, whereas both CMT93 and
CMT93-hsp70 tumors grew initially, in about 50% of the animals the
CMT93-hsp70 tumors regressed compared with 0–10% of the parental
CMT93 tumors (Fig. 1B). This contrasts with 100% regression
of the same tumors in immunocompetent mice (Table I
). Following
surgical excision of these primary tumors and rechallenge with parental
cells on the contralateral flank, the increased immunogenicity of the
B16-hsp70 or CMT93-hsp70 tumors seen in C57BL mice was almost
completely lost in nude mice (Fig. 1, C and D)
compared with that in the control group of naive mice that was
simultaneously challenged with parental tumor cells. In this control
group, tumors were occasionally observed that grew to a small size
(>0.2 cm diameter) but subsequently regressed thereafter. At times, a
small increase in the immunogenicity of B16-hsp70 tumors in nude mice
was observed (Fig. 1C), although this was considerably
reduced relative to the immunogenicity seen in immunocompetent animals
(Table I).
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and GM-CSF
when incubated with parental tumor cells, whereas splenocytes from
naive mice did not (data not shown). However, over several different
experiments, only very low levels of tumor-specific CTL activity (up to
20% specific lysis) could be detected in hsp70-vaccinated mice
relative to that in naive mice or mice vaccinated with B16 cells (data
not shown). This is consistent with our previous experience, in which
it was difficult to detect CTL against B16 cells even when the
protection was clearly mediated by T cells, and with reports from other
groups that antitumor protection does not correlate with CTL levels
(45, 46). Taken together, these data suggest that a major
component of the increased immunogenicity of hsp70-expressing tumor
lines is mediated through T cell involvement. Overexpression of hsp70 in B16 cells does not affect MHC levels or make them more susceptible to NK lysis
Because hsp expression has been described as a target for NK cells (47), we investigated whether the residual antitumor reactivity, not explicable by T cell involvement in the nude mice experiments, was due to NK activity. It has also been reported that hsp expression can increase levels of class I expression (48), which would make the B16-hsp70 cells better targets for T cell recognition in vivo. The parental B16 cells used in this study express very low levels of MHC class I on their surface, but overexpression of the hsp70 gene in B16 cells produced no significant increase in class I expression in the population used for the in vivo studies (data not shown). To test whether expression of hsp70 increased the susceptibility of these low class I B16 cells to NK killing, spleen cells from C57BL mice were cultured with high levels of IL-2 to produce NK (or lymphokine-activated killer) cell populations. Although these NK cells were readily able to lyse NK-sensitive YAC-1 cells, the B16-hsp70 population was, if anything, less susceptible to NK lysis than the parental B16 cells (Fig. 2). Therefore, expression of hsp70 on B16 cells was not able to increase the expression of MHC class I or to increase the sensitivity of parental cells to NK lysis.
Expression of hsp70 induces infiltration of DCs into B16 tumors and intratumoral expression of Th1 cytokines
To investigate which cells other than T cells or NK cells mediate
the antitumor effects of hsp70, the immune infiltrates of B16 and
B16-hsp70 tumors growing progressively in vivo were compared. Tumor
sections stained for a variety of cell surface markers demonstrated
that the only infiltrating cells seen intratumorally in parental B16
tumors were macrophages (Mac3+; Table II). In contrast, a variety of different
immune cells were observed within B16-hsp70 tumors. Consistent with the
results from the nude mice experiments, hsp70-expressing tumors were
infiltrated with both CD4+ and
CD8+ T cells as well as macrophages
(Mac3+). However, the majority of the
tumor-infiltrating leukocytes were Dec205, CD11c+
DCs (Table II and Fig. 3). These DCs were
dispersed through the tumor and clustered near the intratumoral blood
vessels.
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A shows that a weakly hybridizing band
could be detected following RT-PCR for IL-2 within B16 tumors. In
addition, very low levels of mRNA could be detected for IL-10 in B16
tumors, but detection of this cytokine required blotting with an
IL-10-specific probe (data not shown). In contrast, significant
expression of IL-2, TNF-, the p40 subunit of IL-12, and GM-CSF was
detected at relatively high levels in the B16-hsp70 tumors, and weak
expression of IFN- was found, but there was no detectable mRNA for
IL-4, IL-6, or IL-10 (Fig. 4B).
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C).
Consistent with this observation, the B16-hsp70 tumors also showed a
decreased level of expression of tyrosinase relative to B16 tumors
(Fig. 4, A and B). However, B16-hsp70 cells grown
in culture continued to produce melanin at levels comparable to those
produced by B16 cells (data not shown), indicating that the
transfection procedure had not selected out an amelanotic
population. Therefore, expression of hsp70 on B16 tumors induces a dramatic increase in tumor-infiltrating leukocytes comprising T cells, macrophages, and, predominantly, DCs and induces the expression of a Th1-like cytokine within the tumor. Moreover this is accompanied by the apparent loss of melanin production in vivo, which may be due to immunological selection against continued expression of melanocytic Ags.
Protection conferred by hsp70 expression is tumor specific and does not exert a field effect within the tumor
Because B16-hsp70 tumors stimulate such a prominent leukocyte
infiltration, we investigated the mechanism by which its expression
results in antitumor immunity. To test whether the protection conferred
by hsp70 expression was tumor specific, CMT93 cells expressing hsp70
were used as a vaccine against rechallenge with B16 cells in a repeat
of the experiments described in Table I. CMT93-hsp70 tumors gave no
significant protection above that seen with the parental CMT93 line
against rechallenge with B16 cells (data not shown), indicating that
the protection afforded by hsp70 expression is tumor specific.
To test whether the presence of hsp within a tumor containing B16 cells
would be sufficient to immunize against B16, perhaps by a general,
nonspecific, leukocyte-attracting activity, B16 tumors were seeded in
which hsp70 was expressed on 1 x 105 CMT93
cells coseeded within the tumor inoculum. Following surgical excision
of these hybrid tumors, animals were rechallenged on the contralateral
flank with parental B16 cells. Fig.
5 shows that there was no
significant protection conferred by the B16/CMT93-hsp70 hybrid tumors
against B16 rechallenge. These results contrast with those seen when a
smaller number of B16 cells express hsp70 within the vaccinating tumor,
when up to 60% of the animals are protected against rechallenge with
B16 cells (Table I). Therefore, these data suggest that the immune
protection conferred by hsp70 expression is tumor specific and that
hsp70 must be expressed on the tumor cells against which protection is
sought, rather than acting in a diffusable, cytokine-like fashion.
Tumor cell lysates induce maturation of DC precursors, but the presence of hsp maintains the immature phenotype
We investigated whether the presence of hsp70 in tumor cell
lysates would affect the phenotype of the DC precursor populations used
in these studies. As shown in Fig.
6a, the population
of DC precursors had relatively low levels of MHC class I and class II
expression by FACS analysis. Interestingly, when these cells were
incubated with tumor lysates of parental B16 cells, a dramatic
up-regulation of surface MHC expression was observed (Fig. 6c), indicating that tumor cell lysates can induce
maturation of APC. In contrast, incubation of the DC precursors with
lysates from B16-hsp70 cells consistently induced only a very moderate
increase in the level of MHC class I or class II expression (Fig. 6b). These data suggest that lysates from normal tumor cells
may be able to mature DC precursor populations, but that the presence
of hsp may actually suppress these signals.
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The results shown in Figs. 3 and 6 and Table II suggest strongly
that hsp70 expression may exert its immune protective effects through
recruitment and/or modulation of DC function/maturation. Therefore, we
studied the effects of hsp70 expression on DCs and their precursors in
vitro. DC precursors prepared from mouse bone marrow in a low
concentration of GM-CSF (0.2 ng/ml) had a phenotype typical of immature
cells, with relatively low levels of MHC class I and class II
expression as well as other markers, including B7.1 and B7.2 (data not
shown). We and others have shown that these DC precursors can be
matured by culture with higher levels of GM-CSF (20–200 ng/ml) and
even more so with IL-4 to acquire a phenotype with high surface
expression of MHC and costimulatory molecules (data not shown). Fig. 7, A and B, shows that one functional consequence of
the maintenance of the DC precursors in an undifferentiated state, by
growth in low concentrations of GM-CSF and/or incubation with
hsp70-containing tumor cell lysates, is that they remain much more
phagocytic than DC prepared in higher levels of GM-CSF, as assessed by
measuring the uptake of latex beads, rather than shifting the phenotype
to Ag presentation via class I and class II pathways. Consistent with
this hypothesis is the finding that the high GM-CSF-derived cells are
significantly more effective at Ag presentation in an MLR than the low
GM-CSF precursors (data not shown).
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70% protected animals) than is the transfer of more mature DC
(20% protected animals; A. Melcher et al., manuscript in
preparation). Therefore, to test the possibility that hsp70 affects the
functional properties of DC or of their precursors, freshly prepared DC
precursors were incubated with freeze-thawed lysates of B16 or
B16-hsp70 tumor cells that had been metabolically labeled with
[35S]methionine. A significantly enhanced
uptake of labeled proteins was observed in the DC precursors incubated
with hsp70-containing lysates (Fig. 7C). Because our
interest in hsp70 induction and tumor immunogenicity originated from
the observation that hsp70 is induced during HSVtk-mediated
tumor killing, we repeated this experiment using lysates from B16 cells
or B16tk cells that had been killed by ganciclovir (2). A
similar pattern of increased uptake of
35S-labeled proteins, but with a reduced
differential, was seen in the DC precursors incubated with the
GCV-killed B16 cells compared with the parental freeze/thawed lysates
(Fig. 7D). When we repeated these
experiments using DC matured in high GM-CSF concentrations, the ability
of hsp70-containing lysates to increase the uptake of labeled solutes
was completely abolished (data not shown). To investigate the mechanism
of increased Ag capture, these experiments were also repeated either in
the presence of cytochalsin, which is known to block phagocytosis, or
by monitoring the uptake of dextran as a measure of the relative levels
of macropinocytosis. The level of dextran uptake was not affected by
the presence of hsp70 in the tumor lysates, and cytochalasin, an
inhibitor of phagocytosis, was unable to block the increased uptake of
Ags by the DC precursors in the presence of hsp70 (data not shown). Therefore, hsp70 expression on tumor cells specifically targets immature DC precursors and enhances their capacity to capture soluble Ags. Moreover, the increased Ag capture by the DC precursors is not via nonspecific macropinocytosis or phagocytosis and may, therefore, be occurring via a more specific, hsp70 receptor-mediated mechanism.
Hsp70 is taken up into DCs
It may be that the increased levels of uptake of soluble proteins
seen in Fig. 7 represent an indiscriminate activity of hsp70 on DC
precursors to increase their ability to take up Ags or, alternatively,
that the increase is due at least in part to uptake of hsp70 itself.
Therefore, lysates were prepared from the DC precursors that had
themselves been exposed to the radiolabeled tumor lysates as in Fig. 7
and were run on a 10% SDS-PAGE gel. Following transfer to
nitrocellulose the labeled proteins present in the DC were exposed to
film overnight. DC exposed to labeled proteins released from parental
B16 cells showed very low levels of uptake of radiolabel, whereas DC
exposed to lysates from B16-hsp70 cells showed a very significant
increase in uptake of label incorporated into proteins over a range of
m.w. (data not shown), suggesting that no single molecular species is
exclusively taken up by the DC. To investigate whether the hsp70
molecule is itself directly taken up by DC, an expression vector was
constructed in which the hsp70 cDNA was cloned in frame with an Myc tag
peptide (Fig. 8) and transfected into B16
tumor cells. The presence of the tag allowed the movements of the
exogenously expressed hsp70 to be followed and to be distinguished from
those of the endogenous hsp in tumor or DCs. Lysates of DC precursors
preincubated with freeze-thawed lysates of B16 or B16-hsp-Myc cells
were analyzed by Western blot using the anti-Myc tag Ab 9E10. A
specific band at 70 kDa was detected by the Ab in DC precursors
incubated with B16-hsp-Myc cells, but not in DC incubated with B16
cells (Fig. 8B), suggesting that at least one component of
the increased uptake of labeled proteins seen in Fig. 7 is
the hsp70 protein itself.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of hsp70 within B16 tumors induced dramatically increased levels of infiltration by CD4+ and CD8+ T cells, macrophages, and DCs. In addition, hsp70 induced a specific profile of cytokine mRNA expression within the tumor environment that is characteristic of a Th1 immune response (27). Intriguingly, the B16-hsp70 tumors grown in vivo also had greatly down-regulated levels of melanin synthesis and expression of tyrosinase compared with those of the parental tumors. It may be that this reflects the development of hsp70-induced immune responses against melanocytic Ags, which are known to serve as tumor rejection Ags in mouse and human tumors (15), a hypothesis that we are currently testing. However, stimulation of this immune reaction was unable to cause rejection of the primary B16-hsp tumors, presumably because the time required to prime an effective T cell-mediated immune response against B16 is too long relative to the growth of this aggressive tumor in vivo. We had previously observed a very similar immune infiltrate and cytokine profile in B16tk+ tumors treated with GCV (21), which induces hsp70 expression (2). Our results here strongly suggest that the protection produced by HSVtk/GCV killing of B16 tumors is probably mediated at least in part through the induction of hsp70 acting in some way as an immune stimulatory signpost or danger signal.
Our data with T cell-deficient mice demonstrate that hsp70-mediated
protection is predominantly T cell dependent, although CTL could only
be detected at very low levels from splenocytes of animals vaccinated
with hsp70-expressing tumors. In contrast to the up-regulation of MHC
seen in B16 cells by expression of the human hsp72 gene
(48), we did not detect any increase in MHC class I
expression in the B16-hsp70 tumor cells. Previous reports have also
shown that hsp may serve as a target for NK-mediated killing
(47), but we could show no increased susceptibility of
B16-hsp70 cells in vitro. In addition, one component of the protection
induced by expression of hsp70 on tumor cells may be provided by
increased recognition by T cells (37, 38), and we
are currently investigating this possibility. Finally, it may also be
that hsp70 is affecting the expression of an unknown Ag(s) on the B16
cells and promoting enhanced presentation through cross-priming or
other as yet unknown mechanisms.
It may be that expression of hsp70 increases intratumoral infiltrates and leads to T cell-dependent protection by stimulating leukocyte accumulation at the tumor site in a non-cell type-specific manner, in a similar way as a diffusable cytokine, such as GM-CSF or IL-2. Both these cytokines have been shown to have their immunizing effects when delivered by cells other than tumor cells themselves (49, 50, 51). Alternatively, it may be that hsp acts by a cell-associated and tumor cell-specific mechanism (19, 31, 33, 52). In support of the latter, we showed that the protection conferred by hsp70 expression in the B16 model was, indeed, tumor specific, (CMT93-hsp70 tumors were unable to protect significantly against rechallenge with B16 cells). Even within hybrid tumors of B16 and CMT93-hsp70, in which the appropriate (B16) tumor Ags are available, hsp70 must be expressed on the tumor cells against which protection is sought. These observations indicate that hsp70 confers its tumor-immunizing effects in a tumor-specific, cell-associated mechanism rather than acting in a diffusable, cytokine-like fashion.
Therefore, we investigated the effects of hsp70 expression in B16 cells on the form and function of DCs and their progenitors prepared from mouse bone marrow. Interestingly, DC precursors incubated with tumor cell lysates from parental B16 cells consistently demonstrated a significant increase in levels of expression of class I and class II MHC molecules, indicating that tumor lysates can induce maturation of DC precursors. We are currently investigating whether intracellular molecules released during (necrotic) cell death may serve as signals for these effects, as suggested by Matzinger (53). In contrast, the presence of hsp70 inhibited this lysate-induced maturation and maintained the DC precursor population in a more poorly differentiated phenotype. Similarly, we observed a significantly enhanced level of uptake of 35S-labeled proteins by the population of phagocytic DC precursors incubated with hsp70-containing lysates compared with those incubated with parental B16 cell lysates. More mature, less phagocytic DC prepared in high levels of GM-CSF were not responsive to hsp70 in the lysates and showed no increased capacity to take up labeled proteins relative to DC fed parental B16 tumor lysates. Therefore, it appears that hsp70 specifically targets immature DC precursors and enhances their capacity to take up proteins/peptides, but cannot reverse a maturation process already induced by other factors (such as high levels of GM-CSF). Taken together, these data suggest that the maturational status of the DC/DC precursors entering a tumor, which is releasing tumor Ags, may have a significant impact on the efficacy with which the DC can acquire those Ags for subsequent presentation to T cells (54).
A similar enhancement of the protein uptake activity of DC precursors was seen following their exposure to lysates of B16tk+ cells killed in vitro by GCV, indicating that the immune system-activating effects of necrotic HSVtk/GCV killing of B16 cells operate through hsp induction and DC modulation. The net effect would be to increase the ability of tumor-infiltrating DC precursors to take up tumor Ags for later presentation in the tumor or lymph nodes.
A recent report showed that DCs take up Ag released from apoptotic cells, but, in that system, not from cells killed necrotically (18). However, necrosis was induced by rapid lysis ex vivo, with no opportunity for the induction of biochemical/immunological markers of death to be produced. In contrast, the HSVtk/GCV system generates a necrotic death much more akin to that likely to occur in vivo during, for example, a viral infection and occurs over a longer time period during which rapid induction of genes, such as hsp, is possible. Therefore, it is clear that DC can take up Ag from cells under a variety of different conditions (8, 18), but our data suggest that one particularly immune stimulatory pathway is via the induction of hsp expression, which may directly influence the levels of Ag captured for later presentation.
It has been reported that hsp70 recognizes specific receptors on certain cells within the DC precursor population, consistent with a role for an hsp-specific receptor for Ag chaperoning (32) of tumor-specific peptides into APC for class I-mediated presentation (19, 31, 33, 52). These data are also consistent with our finding that hsp70 acts through a cell-associated and tumor specific mechanism. Therefore, we investigated the mechanisms of hsp70 activity by constructing an Myc tag-labeled hsp70 expression vector. These experiments demonstrated that hsp70 is taken up directly by the DC. It is not yet clear what percentage of the increased uptake of labeled proteins is contributed by the hsp70 protein or whether this uptake of hsp70 is directly responsible for increased tumor Ag presentation by a chaperone effect, as reported in other systems (19, 31, 32, 33, 52). However, this hsp70-Myc construct will be valuable to answer these questions and to identify the mechanisms by which hsp70 is taken up into DC, including any receptor that may be involved.
Taken together, our observations suggest a model by which suicide gene killing of tumor cells can, through induction of hsp expression, enhance the immunogenicity of the tumor cells. GCV-mediated killing of tumors can lead to induction of hsp expression in the tumor cells, particularly if the mechanism of dying is by high relative levels of necrotic cell death. This combination of necrotic cell death/induction of hsp signals to the immune system the presence of an immunologically relevant situation against which an immune reaction should be raised. Our data suggest that hsp expression transmits this signal in at least two ways. In the first, accumulation of DCs as well as other immune cells occurs, as evidenced by infiltrates observed within B16-hsp70 tumors. In the second, the presence of hsp within the dying tumor may target immature DC precursors (low class I and II, phagocytic, low Ag-presenting capacity) and further enhance their capacity to capture Ags at a time when large amounts of tumor Ags and cell debris are being released by the HSVtk-GCV-mediated cell killing. In the absence of hsp expression, the nonimmunogenic parental tumor is unable to recruit DC into the tumor for cross-priming, as evidenced by the almost total lack of DC in parental B16 tumor sections. Once the DC have taken up tumor Ag peptides, some of which may be chaperoned into the DC by the hsp itself, they may then complete their maturation in response to additional signals, shifting to a high MHC-expressing phenotype and becoming much more effective at Ag presentation (as confirmed by the high concentration of mature DC (CD11c and Ia +ve cells) observed within the B16-hsp70 tumors). The result is the generation of a T cell-dependent antitumor immune response that can confer long term protection against parental tumors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. Vile, Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; hsp, heat shock protein; HSVtk, HSV thymidine kinase; GCV, ganciclovir.
Received for publication February 17, 1999. Accepted for publication May 19, 1999.
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