Download Express Inducible NKG2D Ligands That Mouse NK Cell Activity

The Heat Shock Protein HSP70 Promotes
Mouse NK Cell Activity against Tumors That
Express Inducible NKG2D Ligands
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of June 17, 2017.
Leslie Elsner, Vijayakumar Muppala, Mathias Gehrmann,
Jingky Lozano, Dörthe Malzahn, Heike Bickeböller, Edgar
Brunner, Marta Zientkowska, Thomas Herrmann, Lutz
Walter, Frauke Alves, Gabriele Multhoff and Ralf Dressel
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:5523-5533; ;
doi: 10.4049/jimmunol.179.8.5523
http://www.jimmunol.org/content/179/8/5523
The Journal of Immunology
The Heat Shock Protein HSP70 Promotes Mouse NK Cell
Activity against Tumors That Express Inducible
NKG2D Ligands1
Leslie Elsner,* Vijayakumar Muppala,* Mathias Gehrmann,† Jingky Lozano,‡
Dörthe Malzahn,‡ Heike Bickeböller,‡ Edgar Brunner,§ Marta Zientkowska,¶
Thomas Herrmann,储 Lutz Walter,# Frauke Alves,¶ Gabriele Multhoff,† and Ralf Dressel2*
T
he stress-inducible heat shock protein (HSP)3 70 is a molecular chaperone which is well known to protect cells
against apoptosis (1). Overexpression of HSP70 has been
described in various tumors and has been found to be associated
with enhanced tumorigenicity and resistance to therapy (2). In accordance with these findings, experimental down-regulation of
HSP70 in tumor cells has been reported to enhance tumor regression in animal models (3–5).
However, in several other animal models contrary observations
have been made, in that HSP70 expression has been found to be
*Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Germany; †Department of Radiotherapy and Radiooncology, Technische Universität München and GSF Institute of Pathology, Munich, Germany; ‡Department of
Genetic Epidemiology, University of Göttingen, Göttingen, Germany; §Department of
Medical Statistics, University of Göttingen, Göttingen, Germany; ¶Department of
Hematology and Oncology, University of Göttingen, Göttingen, Germany; 储Institute
of Virology and Immunobiology, University of Würzburg, Würzburg, Germany; and
#
Department of Primate Genetics, German Primate Center, Göttingen, Germany
Received for publication June 12, 2007. Accepted for publication August 12, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants 10 –1726-Dr I from the Deutsche Krebshilfe (to
R.D.), DR 394/2 from the Deutsche Forschungsgemeinschaft (to R.D.), and MRTNCT-2004 –512253 (TRANS-NET) from the European Union (to R.D., H.B., and
G.M.). V.M. and J.L. were supported by the Research Training Group GRK 1034,
financed by the Deutsche Forschungsgemeinschaft.
2
Address correspondence and reprint requests to Dr. Ralf Dressel, Department of
Cellular and Molecular Immunology, University of Göttingen, Heinrich-Düker-Weg
12, Göttingen. Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: HSP, heat shock protein; HSC, heat shock cognate;
NKG2D, natural killer group 2 member D; MFI, mean fluorescence intensity; MIC,
MHC class I chain-related; SAHA, suberoylanilide hydroxyamic acid.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
associated with tumor regression (6 –9). In these cases, HSP70
appeared to augment the immunogenicity of tumors. In numerous
studies, HSP70 has been shown to activate innate and adaptive
immune reactions (10, 11). HSP70 chaperones antigenic peptides
and channels them, in a receptor-mediated manner, into the MHC
class I presentation pathway of professional APCs, which then
prime peptide-specific CTL (12). Therefore, HSP70 derived from
tumors can be used as tumor-specific vaccines (13). HSP70 also
elicits the release of proinflammatory cytokines from innate immune cells and augments the expression of costimulatory molecules (14, 15). Furthermore, HSP70 has been shown to activate
NK cells to specifically kill tumor cells that express HSP70 at
the cell surface (16). These features have led to HSP70 being
viewed as an endogenous adjuvant and immunological danger
signal (10, 17, 18).
Given that HSP70 is known to be anti-apoptotic but that it can
also elicit a CTL response, we were interested to determine
whether HSP70 protects tumor cells against apoptosis mediated by
CTL. In the model of the human melanoma cell line Ge, we have
shown previously that constitutive overexpression of the MHClinked stress-inducible HSP70 does not protect against apoptosis
mediated by CTL in the granule exocytosis pathway (19). Acute
HSP70 overexpression can even increase the susceptibility against
CTL in vitro (19, 20). The immune system appears to be able to
kill target cells undergoing an otherwise protective stress response.
Our goal was now to determine the effect of HSP70 expression
on the susceptibility of Ge melanoma cells to the cytotoxic effects
of adoptively transferred CTL in vivo. However, in SCID mice
that lack B and T lymphocytes, the growth of HSP70-overexpressing tumors was reduced compared with control tumors, even before any adoptive immunotherapy. More impressively, invasive
growth and regional metastases were only observed in animals
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The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the
immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that
recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that
lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they
killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules
were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B
expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells,
in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells,
which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus,
HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor
growth and suppression of metastatic disease. The Journal of Immunology, 2007, 179: 5523–5533.
5524
HSP70 ACTIVATES MOUSE NK CELLS AGAINST TUMORS
Materials and Methods
Animal experiments
SCID (C.B-17/Ztm-scid) and SCID/beige (C.B-17/IcrHsd-scid-bg) mice
were bred in our own colony under pathogen-free conditions in individually ventilated cages. 129Sv mice serving as spleen donors in some experiments were bred under conventional conditions. The SCID mice were
originally obtained from Dr. H. J. Hedrich (Medizinische Hochschule Hannover, Hannover, Germany) the SCID/beige mice were from Harlan
Winkelmann. Female and male mice between 12 and 20 wk of age were
used for experiments after excluding leaky mice by measuring serum immunoglobulins using an ELISA. All animal experiments had been approved by the local government and were in accordance with institutional
guidelines for the welfare of animals. Tumor cells (1 ⫻ 106 in 100 ␮l PBS)
were injected subcutaneously into the flank of mice. Tumor growth was
monitored every second day by palpation and size was recorded using
linear calipers. Tumor volume was calculated by the formula V ⫽ ␲abc/2,
where a, b, c are the orthogonal diameters. Animals were sacrificed before
a tumor volume of 1 cm3 was reached, when a weight loss of ⬎10%
occurred, or when any behavioral signs of pain or suffering were observable. Autopsies were performed and the abdomen and thoracic cavity were
examined systematically for the presence of metastases. The spleens were
removed for immunological analyses. Parts of primary tumors were immediately frozen in liquid nitrogen for gene expression analyses. Further
tumor and metastatic tissue was placed in phosphate-buffered 4% formalin
for 16 h and then embedded in paraffin. Tissue sections (2.5 ␮m) were
stained with H&E for routine histological examinations. Immunohistochemical staining of the proliferation marker Ki67 was performed as described before (26). For flow cytometric analyses of tumor cells, fresh
tumor tissue was cut into small pieces and incubated in a 5 mg/ml collagenase solution (Sigma-Aldrich) at 37°C for 90 min. Isolated cells were
collected by centrifugation and resuspended in PBS before staining.
Gene expression analysis
RNA was prepared and Northern blots were hybridized and analyzed by
densitometry as described (27). The rat and human MHC-linked inducible
HSP70 proteins are 96.3% identical and 98.4% similar, but they can be
distinguished at the mRNA level by probes specific for the 3⬘ untranslated
region. Thus, hybridization probes specific for the MHC-linked rat
Hsp70-1 (positions 2875 to 3070; accession no. X77207) (28) and the
orthologous human HSP70-2 gene (positions 2225 to 2407, accession no.
M59830) (29) were derived from the 3⬘ untranslated region of the respective genes by genomic PCR amplification. A MICA gene probe encompassing exons 2 to 5 (30) was used to detect MICA and MICB transcripts,
which can be distinguished by size. The human ␤-actin cDNA was purchased from Clontech.
Target cell lines and cell culture
The human melanoma cell line Ge, the Ge-Hsp70 and Ge-con sublines
derived therefrom (clones Ge-Hsp70-A, Ge-Hsp70-C, Ge-TCR-C, and GeGFP-B) (19), human erythroleukemia K562 cells, and mouse lymphoma
YAC-1 cells were maintained at 37°C in NaHCO3-buffered DMEM sup-
FIGURE 1. Tumors derived from HSP70-overexpressing melanomas
grow slower and do not give rise to metastases. a, Ge-Hsp70 or Ge-con
cells were injected s.c. into the flank of SCID mice (1 ⫻ 106 cells in
PBS/animal). Representative photographs are shown. The black arrow (I)
points toward a noninvasively grown s.c. tumor (II). Invasive growth (III)
was only observed for control tumors. Metastases in the mesenterium (IV),
in an axillary lymph node (V, black arrow), and in the diaphragm (VI) after
injection of Ge-con cells are shown. b, The mean of tumor size ⫾ SD for
SCID mice in which tumor growth was observed after injection of GeHsp70 or Ge-con cells is shown.
plemented with 10% FCS (Biochrom), 2 mM L-glutamine, 1 mM sodium
pyruvate, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (SigmaAldrich). To induce MICA/B expression, Ge cells were cultured in DMEM
with 10 ␮M of the histone deacetylase inhibitor suberoylanilide hydroxyamic
acid (SAHA) (Qbiogene-Alexis) 20 h before the tests (31). The heat shock
treatment of melanoma cells was performed as previously described (19).
Proliferation and apoptosis of Ge melanoma cells
To analyze in vitro proliferation 2 ⫻ 104 cells per well were seeded in 200
␮l DMEM in micro titer plates for cell culture (Sarstedt) in triplicates for
each time point of measurement (12, 24, 48, and 72 h). Twelve hours
before harvesting 1 ␮Ci [methyl-3H]thymidine (specific activity 5 Ci/
mmol, Amersham) was added to the respective wells. Triplicates were
harvested using a Titertek cell harvester 550 (Flow Laboratories). Incorporated radioactivity was determined by liquid scintillation counting using
a Wallac MicroBeta Trilux counter (PerkinElmer Life Sciences). Apoptosis
was induced by hypoxia and glucose starvation. To expose cells to hypoxic
conditions, petri dishes were placed in a GasPak 100 system (BD Biosciences). After 2 h the O2 concentration in the system is ⬍1% and the CO2
concentration reaches 10% (32). The GasPak system was placed for 24 h
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
bearing non-HSP70-overexpressing control tumors. We show in
this study that the stress-inducible danger signal HSP70 activates
mouse NK cells in SCID mice which recognize inducible NKG2D
ligands on tumor cells. NKG2D (NK group 2, member D) is an
activating NK receptor (21). The MHC class I chain-related (MIC)
A and B molecules are NKG2D ligands in humans (21) and their
expression was induced in the human tumors in SCID mice. MICA
and MICB genes are encoded within the MHC, are stress inducible,
and are expressed in a restricted manner in intestinal epithelial
cells and in tumors (22). Although the MICA and MICB genes are
not present in the mouse genome, the human molecules functionally interact with mouse NKG2D (23–25). In mice, the retinoic
acid early inducible-1 (Rae-1), and UL16-binding protein-like
transcript 1 (Mult1) gene products and a minor histocompatibility
Ag (H60) have been reported as NKG2D ligands (21). The inducible HSP70 and inducible NKG2D ligands, which both appear to
function as immunological danger signals, synergistically elicited
a NK cell-mediated immune response against tumor cells. This two
danger signals-driven innate immune response was able to reduce
the growth of primary tumors and suppress metastatic disease.
The Journal of Immunology
5525
Table I. Tumor frequency and formation of metastases after subcutaneous inoculation of Ge-Hsp70 or
Ge-con cells into SCID and SCID/beige mice
Inoculated cellsa
SCID Mice
Ge-Hsp70
Ge-Hsp70-A
Ge-Hsp70-C
Ge-con
Ge-TCR-C
Ge-GFP-B
Ge
SCID/beige mice
Ge-Hsp70
Ge-Hsp70-A
Ge-Hsp70-C
Ge-con
Ge-TCR-C
Ge-GFP-B
Tumor frequency
(day 24)b
Tumor frequency
(at autopsy)c
Metastases
(at autopsy)d
73 % (30/41)
73 % (16/22)
74 % (14/19)
86 % (38/44)
87 % (20/23)
86 % (18/21)
76 % (35/46)
73 % (30/41)
73 % (16/22)
74 % (14/19)
86 % (38/44)
87 % (20/23)
86 % (18/21)
85 % (39/46)
0 % (0/30)
0 % (0/16)
0 % (0/14)
21 % (8/38)
25 % (5/20)
17 % (3/18)
18 % (7/39)
88 % (22/25)
92 % (11/12)
85 % (11/13)
92 % (22/24)
84 % (10/12)
100 % (12/12)
88 % (22/25)
92 % (11/12)
85 % (11/13)
96 % (23/24)
92 % (11/12)
100 % (12/12)
23 % (5/22)
18 % (2/11)
22 % (3/11)
22 % (5/23)
27 % (3/11)
17 % (2/12)
in an incubator at 37°C. For glucose starvation the melanoma cells were
cultured for 24 h in glucose-free DMEM (Sigma), which was supplemented
as standard DMEM. Propidium iodide positive dead cells and apoptotic
cells appearing in the sub G1 peak of DNA histograms were determined as
described previously (27).
Preparation of exosomes and immunoblot analysis
The Ge-Hsp70 and Ge-con cell lines were grown to ⬃80% confluence
before being cultured in fresh DMEM for 72 h. Cell viability was ⬎95%,
as determined by trypan blue exclusion. The supernatant was harvested and
exosomes were prepared as described (33) and analyzed by SDS-PAGE.
Immunoblotting was performed (33) using Abs specific for the inducible
form of HSP70 (mAb C92, clone C92F3A-5, mouse IgG1, SPA-810,
StressGen; Biomol) and against Rab4 (sc-312, rabbit Ab; Santa Cruz
Biotechnology).
Effector cells, effector cell culture and
assays
51
Chromium release
Splenocytes from mice were obtained using a Tenbroeck homogenizer and
erythrocytes were removed by incubation for 5 min in lysis buffer (155 mM
NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.4 –7.8)). The cells obtained
from tumor bearing mice were used either directly as cytotoxic effector
cells or cultured for 24 h in DMEM with 10% FCS and 20% supernatant
from Con A-stimulated lymphocytes before being used in 51Chromium
release assays. To stimulate NK cells in vitro, splenocytes from SCID or
immunocompetent 129Sv mice were cultured for 2 days in 5-ml petri
dishes for tissue culture (Sarstedt) at a density of 2 ⫻ 107 cells/ml in
DMEM supplemented with 10% FCS, 20% supernatant from Con A-stimulated lymphocytes, 50 ␮M 2-ME (Sigma-Aldrich), and 20 ng/ml mouse
IL-2 (Immunotools). To some of these cultures exosomes prepared from
Ge-con or Ge-Hsp70 cells were added at a concentration of 10 ␮g/ml. NK
cells were isolated after two days of culture by MACS using a negative
selection kit (NK cell isolation Kit mouse, 130-090-864; Miltenyi Biotec).
The kit contains a mixture of Abs against CD4, CD5, CD8a, CD19, Ly-6G,
and Ter-119 and depletes the non-NK cells. 51Chromium release assays
were performed and the percentage of specific cell lysis was determined as
previously described (27). A recombinant mouse NKG2D-Fc chimeric protein (139-NK) purchased from R&D Systems was used to inhibit the part
of lysis of target cells which was dependent on the expression of NKG2D
ligands. The recombinant protein was added at a concentration of 3 ␮g/ml
to 51Chromium release assays.
Flow cytometry
Flow cytometry was performed on a FACScan flow cytometer (BD Biosciences) using CellQuest data acquisition and analysis software. Expres-
sion of intracellular HSP70, TCR␤, and GFP in Ge-Hsp70 and Ge-con
clones, respectively, was regularly monitored by flow cytometry as described previously (19). Cell surface expression of HSP70 on propidium
iodide negative cells was examined using a mAb which has been reported
(34, 35) to detect HSP70 on the plasma membrane (RPN 1197, mouse
IgG1, Amersham Pharmacia). MICA/B cell surface expression was determined using the BAMO-1 mAb which reacts with human MICA and
MICB (mouse IgG1; Immatics). The W6/32 mAb (mouse IgG1, Serotec)
was used for staining of cells for human MHC class I molecules. The
binding of these unlabeled mouse IgG Abs was revealed using polyclonal
FITC-conjugated goat anti-mouse IgG Abs (115-095-062; Jackson Laboratories). A recombinant mouse NKG2D-Fc chimeric protein (139-NK)
was used to detect cell surface expression of NKG2D ligands. For these
experiments polyclonal FITC-conjugated goat anti-human IgG Abs (109095-098; Jackson Laboratories) were used as secondary reagent. The percentage of NK cells in the spleens and tumors of SCID mice was determined using the pan-NK cell marker CD49b (clone DX5, rat IgM, PEconjugated; Caltag Laboratories). NK cell enriched and depleted fractions
from splenocyte cultures were characterized using the anti-CD49b Ab
and Abs reactive against CD3 (clone 145-2C11, hamster IgG, FITCconjugated, Immunotools), CD4 (clone CT-CD4, rat IgG2a, tricolorconjugated; Caltag Laboratories), CD8a (clone CT-CD8a, rat IgG2a,
PE-conjugated; Caltag Laboratories), and CD19 (clone 6D5, rat IgG2a,
FITC-conjugated; Caltag Laboratories). Isotype controls (rat IgG2a, rat
IgM, and hamster IgG) were purchased from Caltag Laboratories.
Statistics
All data were analyzed using the SAS version 9.1 software. ANOVA was
used to analyze designs involving two or more factors. The different factors
were incorporated into a two-way or three-way ANOVA involving interactions. The t test was used for the analysis of paired and unpaired two
samples. A repeated measures ANOVA was conducted in all experimental
designs having replicates, such as 51Chromium release assays. A significance level of ␣ ⫽ 0.05 was used. Adjustments for multiple comparisons
were performed where appropriate.
Results
Reduced tumor growth of HSP70-overexpressing melanoma cells
The human melanoma cell line Ge was retrovirally transduced to
constitutively overexpress the normally stress-inducible MHClinked rat Hsp70-1 (Hspalb) gene. Control cell clones (Ge-con)
were obtained by transduction with a rat TCR␤-chain or GFP expression construct derived from the same vector. Both the GeHsp70 and the Ge-con clones have been previously described and
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a
Ge-Hsp70 cells (clones Ge-Hsp70-A and Ge-Hsp70-C), Ge-con cells (clones Ge-TCR-C and Ge-GFP-B), as well as
parental Ge melanoma cells were injected subcutaneously into the flank of SCID or SCID/beige mice (1 ⫻ 106 cells in
PBS/animal).
b
Percentage and number of animals in which primary tumors were palpable at day 24, before the first mice had to be
sacrificed.
c
Percentage and number of animals in which primary tumors were palpable at the time point of section (day 26 to day 36).
d
Tumor-bearing animals were carefully inspected for metastases during autopsy and the percentage and number of animals
with metastases is given.
5526
HSP70 ACTIVATES MOUSE NK CELLS AGAINST TUMORS
No effect of constitutive overexpression of HSP70 on
proliferation and apoptosis
Staining of tumors with the proliferation marker Ki67 did not reveal major differences between Ge-Hsp70 and Ge-con-derived tumors (data not shown). The in vitro proliferation of HSP70-overexpressing and control clones, as determined by [3H]thymidine
incorporation and cell counting was similar (data not shown).
Thus, the reduced growth of HSP70-overexpressing tumors could
not be explained by differences in the proliferation rate. Therefore,
we analyzed cell death and apoptosis after exposure of the cells to
conditions that occur in tumors, such as hypoxia and glucose starvation. Again, no difference between the Ge-Hsp70 and the Ge-con
cells was observed when apoptosis was assessed by sub G1 peak
measurement or cell death by propidium iodide staining after exposure to hypoxia or glucose-free medium for 24 h (data not
shown).
Augmented cellular cytotoxicity in SCID mice bearing
HSP70-overexpressing tumors
FIGURE 2. NK cells are responsible for the reduced growth of HSP70overexpressing melanomas in SCID mice. a, The mean percentage of NK
cells in the spleen ⫹ SD, detected by flow cytometry using the anti-CD49b
mAb DX5 is shown for SCID mice with Ge-con or Ge-Hsp70 tumors or
animals which rejected the tumor cells (no tumors). b, The mean percentage of tumor infiltrating NK cells ⫹ SD in Ge-Hsp70 and Ge-con tumors
is shown. Freshly isolated cells were stained using the anti-CD49b mAb
DX5 and analyzed by flow cytometry. c, The mean specific lysis ⫾ SD of
triplicates of YAC-1 target cells by splenocytes derived from three SCID
mice bearing Ge-con tumors, three SCID mice bearing Ge-Hsp70 tumors,
and two tumor-free SCID mice at different E:T ratios is shown. The
We then speculated that the innate immune system, which is still
present in SCID mice, might contribute to the partial control of the
growth of HSP70-overexpressing melanomas. To address this
question, the number and the activity of cytotoxic cells from mice
bearing HSP70-overexpressing or control tumors was analyzed.
The percentage of splenic NK cells of mice which rejected tumors
and mice in which Ge-Hsp70 or Ge-con tumors grew did not differ
significantly (Fig. 2a). Moderate numbers of NK cells were found
in the established tumors by flow cytometric analysis of freshly
prepared cell suspensions. They were present in a slightly higher
frequency ( p ⫽ 0.0081, t test) in Ge-Hsp70 than in Ge-con tumors
at the time point of autopsy (Fig. 2b). More interestingly, the cytotoxic activity of splenocytes from mice with HSP70-overexpressing tumors against the NK cell sensitive target cell line
experiment is representative of five independent assays. d, Ge-Hsp70 or
Ge-con cells were injected subcutaneously into the flank of SCID/beige
mice (1 ⫻ 106 cells in PBS/animal). The mean of tumor size ⫾ SD for
SCID mice in which tumor growth was observed is shown.
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characterized in detail by in vitro analyses (19). Two clones of the
Ge-Hsp70 cells (Ge-Hsp70-A and Ge-Hsp70-C) and two clones of
the control cells (Ge-TCR-C and Ge-GFP-B) were selected for this
study. The expression of HSP70 and control proteins in these
clones was controlled regularly (data not shown) and found to be
stable and in the range which we have previously described (19).
Ge-Hsp70 and Ge-con cells were injected subcutaneously into the
flank of SCID mice, which lack T and B lymphocytes. The primary
tumors grew progressively and at day 26 the first animals had to be
sacrificed (Fig. 1a). Surprisingly, the growth of HSP70-overexpressing tumors was reduced compared with control tumors. The
frequency of tumors (Table I) on day 24 was slightly decreased in
animals injected with Ge-Hsp70 cells (73%) compared with animals injected with Ge-con cells (86%). Most tumors grew locally
(Fig. 1a, I and II) but in several cases invasive growth of the
primary tumors (III) and regional metastases in the mesenterium
(IV) were observed. Some metastases were additionally found in
regional lymph nodes (V) and in the diaphragm (VI). Intriguingly,
the frequency of metastases was 21% for the Ge-con and 18% for
the parental Ge tumors, whereas none of the Ge-Hsp70 tumors
gave rise to metastases (Table I). Thus, overexpression of HSP70
appears to reduce the malignancy of the Ge melanoma cells. Furthermore, even when tumors developed from Ge-Hsp70 cells, their
growth rate was significantly reduced ( p ⫽ 0.0039, ANOVA)
compared with tumors derived from Ge-con cells (Fig. 1b).
The Journal of Immunology
5527
YAC-1 was augmented when compared with mice with control
tumors or tumor-free mice (Fig. 2c). This result suggests an increased cellular cytotoxic activity in SCID mice bearing HSP70overexpressing tumors. Although the in vitro cytotoxic activity of
splenocytes derived from SCID mice against the Ge-Hsp70 and
Ge-con cells was generally low (data not shown), these results
pointed toward a role of cytotoxic cells present in SCID mice, e.g.,
NK cells, in controlling the growth of Ge-Hsp70 tumors.
Tumor growth of HSP70-overexpressing melanoma cells is not
reduced in SCID/beige mice
To verify the suspected role of NK cells in vivo, we injected the
same cell clones as before into SCID/beige mice which lack functional NK cells in addition to T and B lymphocytes. The comparative analysis of tumor growth in SCID and SCID/beige mice has
been widely used to determine the role of NK cells in tumor regression (36 – 40). The frequency (Table I) and the growth of tumors (Fig. 2d) after injection of Ge-Hsp70 and Ge-con cells were
similar in these animals. Tumors derived from Ge-Hsp70 cells
resulted in the same metastatic frequency as tumors from Ge-con
cells (Table I). Furthermore, splenocytes from tumor-bearing
SCID/beige mice did not kill YAC-1 cells efficiently in vitro irrespective of the tumor type that was present in the mice (data not
shown). These results clearly indicate that the differences observed
in the SCID mice were due to the activity of NK cells, that NK
cells partially control the growth of Ge-Hsp70-derived tumors and
that they completely suppress metastases.
No HSP70 but MICA/B cell surface expression on tumors
It has been shown previously that HSP70 which is expressed at the
cell surface of some tumor cells can function as a target structure
for NK cells (16). Thus, we analyzed the expression of HSP70 on
the melanoma cells using an Ab suitable for HSP70 cell surface
staining (35). Cultured Ge-Hsp70 and Ge-con cells were negative
for plasma membrane-associated HSP70 (data not shown). Cells
obtained from freshly prepared tumors from SCID or SCID/beige
mice also did not express cell surface HSP70 (Fig. 3a), although
the transgenic rat Hsp70-1 mRNA was strongly expressed in the
Ge-Hsp70-derived tumors (Fig. 3b) and some stress-inducible endogenous human HSP70-2 mRNA was detectable in all tumors
(Fig. 3c). Therefore, we next asked whether other ligands for activating NK receptors might be present on the tumors. MICA and
MICB molecules were among the candidates, because these human
ligands have also been shown to interact structurally and functionally with the mouse activating NK receptor NKG2D (23–25). Expression of MICA and MICB mRNA in Ge-Hsp70, Ge-con and
parental Ge cells was very low in vitro (Fig. 4, a and b). In tumors,
however, the expression of both MIC genes was clearly induced
(Fig. 4, a and b). Interestingly, MICA and MICB mRNA expression in tumors grown in SCID/beige was higher than that in SCID
mice (MICA: p ⫽ 0.0146 and MICB: p ⬍ 0.0001, ANOVA).
Furthermore, we observed a differential expression of MICB
mRNA in Ge-Hsp70 vs Ge-con tumors that was clearly modified
by the host. MICB mRNA expression was lower in Ge-Hsp70 than
Ge-con tumors grown in SCID mice (interaction: p ⫽ 0.0437,
ANOVA), whereas such a difference was not found in SCID/beige
mice. A similar tendency for MICA mRNA expression was observed, although not on a statistically significant level. In subsequent experiments, MICA/B molecules were confirmed to be expressed at the cell surface of tumor cells in vivo by flow cytometry
after staining single cell suspensions derived from fresh tumors
with an anti-MICA/B mAb or recombinant mouse NKG2D (Fig.
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FIGURE 3. HSP70 is not expressed on the cell surface of tumor cells ex vivo despite strong expression of transgenic rat Hsp70-1 and modest induction
of endogenous human HSP70-2 mRNA in tumors. a, A flow cytometric analysis of HSP70 cell surface expression (mAb RPN 1197) on cells freshly isolated
from Ge-con and Ge-Hsp70 tumors is shown. The percentage of positive cells is given. The experiment is representative for eight tumors from SCID and
ten from SCID/beige mice. K562 cells served as positive control. b, Mean of mRNA expression ⫹ SD of transgenic rat Hsp70-1 determined as ratio to
␤-actin by Northern blot analysis and densitometry. c, Mean of mRNA expression ⫹ SD of endogenous human HSP70-2 determined as ratio to ␤-actin
by Northern blot analysis and densitometry. For b and c, 18 Ge-con and 21 Ge-Hsp70 tumors from SCID mice and 16 Ge-con and 14 Ge-Hsp70 tumors
from SCID/beige mice were analyzed. The data for the cell lines in vitro were obtained from 7 to 12 individual experiments. Note the different scales in
the diagrams b and c.
5528
HSP70 ACTIVATES MOUSE NK CELLS AGAINST TUMORS
4c). In accordance with the mRNA expression data, a reduced
MICA/B cell surface expression was observed on Ge-Hsp70 tumors grown in SCID mice ( p ⫽ 0.002, t test) compared with
Ge-Hsp70 tumors grown in SCID/beige hosts (Fig. 4d). Furthermore, the MICA/B expression was different in Ge-Hsp70 and Gecon tumors grown in SCID ( p ⫽ 0.0048, t test) but not in those
grown in SCID/beige mice. These data suggest a functional role of
MICA/B expression in the Ge-Hsp70 tumors. We assume that in
SCID mice tumor cells which express MICA/B become targets for
NK cells. Therefore, a selection against MICA/B expressing tumor
cells in SCID but not in NK cell deficient SCID/beige mice might
have occurred. This selection pressure was more apparent for
HSP70-overexpressing tumors than for control tumors, perhaps
due to a higher cytotoxic activity of NK cells in mice with HSP70overexpressing tumors.
Augmented cellular cytotoxicity in SCID mice with
HSP70-overexpressing tumors depends on the expression
of NKG2D ligands on target cells
Splenocytes from SCID mice with HSP70-overexpressing tumors
were able to lyse YAC-1 target cells (Fig. 2c). However, in vitro
they did not lyse the human Ge melanoma cells (data not shown).
This low cytotoxic activity against the Ge target cells might be
explained by the fact that MICA/B molecules were not expressed
in these cells under normal cell culture conditions. YAC-1 cells are
a typical target cell line for murine NK cells and they express
constitutively high amounts of NKG2D ligands in contrast to the
human Ge melanoma cells (Fig. 5a). However, NKG2D ligands
were inducible on the melanoma cells by treatment with the histone deacetylase inhibitor SAHA (Fig. 5a) that is known to induce
MICA/B (31). Splenocytes from naive, tumor-free SCID mice did
not kill YAC-1 or Ge melanoma cells irrespective of the expression of NKG2D ligands (Fig. 5b). Splenocytes from SCID mice
with HSP70-overexpressing tumors (Ge-Hsp70) were able to lyse
YAC-1 cells and SAHA-treated Ge-con or Ge-Hsp70 cells, which
expressed NKG2D ligands but not untreated melanoma cells
which were negative for NKG2D ligands (Fig. 5, c and d). Splenocytes from SCID mice with control tumors (Ge-con) had a much
lower cytotoxic activity and killed even YAC-1 cells only inefficiently (Fig. 5, c and d). These findings suggest a role of tumorderived HSP70 in the activation of NK cells which subsequently
recognize NKG2D ligands on target cells.
No regulation of MICA/B expression by HSP70 overexpression
To explain the role of HSP70 in the tumors, one might alternatively speculate that overexpression of HSP70 as a molecular
chaperone could allow for an increased cell surface expression of
MICA/B molecules which subsequently augment the sensitivity of
HSP70-overexpressing tumor cells to NK cells. However, in vivo
the expression of MICA/B was not different in Ge-con and GeHsp70 tumors grown in SCID/beige mice (Fig. 4). Also in vitro the
expression of MICA/B on Ge-con and Ge-Hsp70 cells was similar
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FIGURE 4. MICA/B expression is induced in tumors but the expression level depends on the NK cell activity in the host. a, The mean of mRNA
expression ⫹ SD of MICA was determined as ratio to ␤-actin by Northern blot analysis and densitometry. b, The mean of mRNA expression ⫹ SD of MICB
was determined as ratio to ␤-actin by Northern blot analysis and densitometry. For a and b, 18 Ge-con and 21 Ge-Hsp70 tumors from SCID mice and 16
Ge-con and 14 Ge-Hsp70 tumors from SCID/beige mice were analyzed. The data for the cell lines in vitro were obtained from 7 to 12 individual
experiments. The rat Hsp70-1 and human HSP70-2 mRNA expression in the same set of samples is shown in Fig. 4, b and c. c, The cell surface expression
of MICA/B (mAb BAMO-1) and NKG2D ligands (mouse NKG2D-Fc chimeric protein) was analyzed by flow cytometry on cells isolated from a Ge-con
tumor grown in a SCID mouse. The staining using the specific mAb plus secondary Ab (black line) and the secondary Ab alone (dotted line) is shown.
More than 95% of the cells gated in forward/side scatter plots according to tumor cell characteristics were positive for human MHC class I molecules (mAb
W6/32). d, The mean ⫹ SD of the specific MFI (MFI for the specific staining minus MFI for the staining with the secondary Ab alone) is shown for MICA/B
cell surface staining (mAb BAMO-1) on freshly isolated cells from Ge-con or Ge-Hsp70 tumors grown in SCID or SCID/beige hosts (n ⫽ 8 for each group).
The Journal of Immunology
5529
(Fig. 6a). MICA/B expression was also inducible to the same extend on Ge-con and Ge-Hsp70 cells (Fig. 6a) by the histone
deacetylase inhibitor SAHA. Induction of intracellular HSP70 in
Ge-con and Ge-Hsp70 cells by heat shock (Fig. 6b) was also not
accompanied by an induction of MICA/B molecules (Fig. 6a).
Moreover, no synergistic effect of heat shock and SAHA treatment
on MICA/B cell surface expression was observed (Fig. 6a). Thus,
neither constitutive nor induced HSP70 overexpression did affect
the expression of MICA/B on the melanoma cells, arguing strongly
against an epistatic regulation of MICA/B expression by HSP70.
HSP70 containing exosomes are released from
HSP70-overexpressing melanoma cells
The function as target structure for NK cells (16) is not the only
role that has been assigned to HSP70 in NK cell biology. Extracellular HSP70 has been shown to activate NK cells (41). Therefore, we determined whether HSP70 is released from the melanoma cells. It is known that cells, including tumor cells, can
release exosomes which contain heat shock proteins (33, 42). In
contrast to Ge-con cells, viable Ge-Hsp70 cells did release exosomes containing the inducible HSP70 (Fig. 7a). These findings
indicate that the HSP70-overexpressing melanoma cells are a
source for extracellular HSP70 that might activate NK cells.
HSP70-positive exosomes activate mouse NK cells to kill
NKG2D ligands expressing target cells
Taken together, our findings suggest that HSP70 is released via
exosomes from the HSP70-overexpressing tumor cells and activates NK cells which then kill preferentially the MICA/B expressing human melanoma cells. To further test this hypothesis, splenocytes from SCID mice were cultured in the presence of IL-2 (20
ng/ml) or IL-2 plus exosomes (10 ␮g/ml) derived from Ge-con or
Ge-Hsp70 cells. After two days NK cells were negatively isolated
by MACS and used as effector cells in a 51Chromium release assay
against YAC-1, Ge-con, and SAHA-treated Ge-con target cells
(Fig. 7b). The HSP70-positive exosomes of Ge-Hsp70 cells in contrast to exosomes of Ge-con cells were able to stimulate the cytotoxic activity of NK cells against YAC-1 and SAHA-treated Gecon cells. Untreated Ge-con cells, which did not express NKG2D
ligands, remained resistant to lysis (Fig. 7b). The NK cell depleted
fractions of the splenocyte cultures were not able to kill any of the
targets even at higher E:T ratios (data not shown). A summary of
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FIGURE 5. Splenocytes from mice with Hsp70-overexpressing tumors are activated to lyse tumor cells which express NKG2D ligands. a, The cell
surface expression of NKG2D ligands was analyzed by flow cytometry on YAC-1, Ge-con, and Ge-Hsp70 cells using a recombinant mouse NKG2D-Fc
fusion protein. Staining with the primary reagent (black line) and FITC-labeled secondary reagent only (dashed line) is shown together with unstained cells
(dotted line). The melanoma cells were either cultured under standard conditions or exposed to the histone deacetylase inhibitor SAHA (10 ␮M) for 20 h
before the test. The results are representative for ⬎5 independent experiments. b, The mean specific lysis ⫾ SD of triplicates of target cells by splenocytes
derived from a naive, tumor-free SCID mouse at different E:T ratios is shown. Target cells are YAC-1, Ge-con, and Ge-Hsp70. The Ge cells were also tested
after exposure to 10 ␮M SAHA for 20 h before the test to induce NKG2D ligands. c, The mean specific lysis ⫾ SD of triplicates of target cells by
splenocytes derived from a SCID mouse with a Ge-con tumor and a SCID mouse with a Ge-Hsp70 tumor at different E:T ratios is shown. Target cells are
YAC-1, Ge-con, and Ge-con exposed to 10 ␮M SAHA for 20 h before the test to induce NKG2D ligands. d, The mean specific lysis ⫾ SD of triplicates
of target cells by splenocytes derived from a SCID mouse with a Ge-con tumor and a SCID mouse with a Ge-Hsp70 tumor at different E:T ratios is shown.
Target cells are YAC-1, Ge-Hsp70, and Ge-Hsp70 exposed to 10 ␮M SAHA for 20 h before the test to induce NKG2D ligands.
5530
three independent experiments with NK cells from 129Sv mice is
shown in Fig. 8a, indicating that also NK cells from normal immunocompetent mice can be stimulated by exosomes of Ge-Hsp70
cells, in contrast to exosomes of Ge-con cells, to kill target cells
which express NKG2D ligands such as YAC-1 ( p ⬍ 0.05,
ANOVA) and SAHA-treated Ge-con cells ( p ⬍ 0.05, ANOVA).
Similar results were obtained with Ge-Hsp70 instead of Ge-con
cells as targets (data not shown). The proportion of CD49b (DX5)positive NK cells obtained after stimulation with IL-2 only or IL-2
plus exosomes derived from Ge-con or Ge-Hsp70 cells was not
different (Fig. 8b). The proportion of contaminating B and T lymphocytes was always below 5% (data not shown). The lysis of
YAC-1 and SAHA-treated Ge-con cells could be inhibited by a
soluble mouse NKG2D-Fc chimeric protein as exemplified in Fig.
8c. This finding suggests that the killing was indeed dependent on
the recognition of NKG2D ligands on the target cells. Thus, the
HSP70 containing exosomes appear to augment the cytotoxic activity of the NK cells against target cells which express NKG2D
ligands.
Discussion
The stress response is destined to maintain survival of cells that
have been exposed to adverse environmental conditions. To be
successful the immune system must have the capacity to destroy
target cells, even during an ongoing stress response which has been
initiated for cellular protection. Moreover, stressed cells, e.g., virus-infected cells or tumor cells, appear to be usually a more appropriate target for cytotoxic effector cells of the immune system
than unstressed cells (43). We have previously shown that HSP70,
which is known to protect cells efficiently against various adverse
FIGURE 7. HSP70-positive exosomes are released from Ge-Hsp70
cells and can stimulate in vitro the cytotoxic activity of NK cells from
SCID mice to kill target cells which express NKG2D ligands. a, Exosomes
of Ge-Hsp70 and Ge-con cells were prepared, 10 ␮g of exosomal proteins
were separated by SDS-PAGE and analyzed for the presence of HSP70 by
immunoblot using the mAb C92 which is specific for the inducible HSP70.
An anti-Rab4 Ab was used as loading control for the exosomal proteins. b,
The mean of specific lysis ⫹ SD of target cells by NK cells from SCID
mice at different E:T ratios are shown as determined in a representative
experiment. YAC-1, Ge-con, and Ge-con cells exposed to 10 ␮M SAHA
for 20 h before the tests served as targets. NK cells were isolated by MACS
from splenocytes of naive SCID mice after stimulation in vitro for 2 days
with IL-2 (20 ng/ml) plus exosomes (exo) of Ge-con cells (10 ␮g/ml), or
IL-2 plus exosomes of Ge-Hsp70 cells. The proportion of CD49b (DX5)positive NK cells among the effector cells were 73% after stimulation with
exosomes of Ge-con cells and 69% after stimulation with exosomes of
Ge-Hsp70 cells. The MICA/B induction on the Ge-con cells by SAHA
treatment was in the same range as shown in Fig. 6a.
conditions, fails to protect against specific cytotoxic effector mechanisms of CTL mediated in the granule exocytosis pathway (19,
20, 27). Thus, cytotoxic effector mechanisms of the cellular immune system seem to dominate over the protective stress response.
In accordance with this assumption, it has been suggested that
components of the stress response system function as endogenous
danger signals which trigger the initiation of an immune response
(17, 18). HSP70, e.g., appears capable of activating and connecting
innate and adaptive immune reactions (10, 18), and fulfills the
criteria of endogenous danger signals. Further examples of potential endogenous danger signals are the ligands of the NKG2D receptor (44). NKG2D has been shown to serve as an activating
receptor triggering NK cell responses against tumors and expression of NKG2D ligands in tumors has been reported to induce
tumor rejection (22, 45, 46). NKG2D ligands include in humans
MICA and MICB. These NKG2D ligands appear to be up-regulated in response to stress (21, 22, 47, 48), and they signal the
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FIGURE 6. MICA/B expression is not regulated by HSP70. a, Ge-con
and Ge-Hsp70 cells were cultured under standard conditions (co), treated
with the histone deacetylase inhibitor SAHA (10 ␮M) for 24 h, heat
shocked (HS, 1 h at 42°C plus 23 h at 37°C), or treated with SAHA and
heat shock before flow cytometric analysis of MICA/B cell surface expression (mAb BAMO-1). The mean ⫹ SD of the specific MFI (MFI for the
specific staining minus MFI for the staining with the secondary Ab alone)
of six experiments with Ge-con and seven with Ge-Hsp70 cells is shown.
b, The same cells were analyzed in parallel for intracellular HSP70 expression (mAb C92) by flow cytometry. The mean ⫹ SD of the specific
MFI is shown.
HSP70 ACTIVATES MOUSE NK CELLS AGAINST TUMORS
The Journal of Immunology
5531
FIGURE 8. NK cells from immunocompetent mice are stimulated by
exosomes of Ge-Hsp70 cells to kill target cells which express NKG2D
ligands. a, The mean of specific lysis ⫹ SD of target cells by NK cells from
129Sv mice at different E:T ratios is shown as determined in three independent experiments. In the individual experiments triplicates of the target
cells were analyzed. YAC-1, Ge-con, and Ge-con cells exposed to 10 ␮M
SAHA for 20 h before the tests served as targets. The MICA/B induction
on the Ge-con cells by SAHA treatment was in the same range as shown
in Fig. 6a. NK cells were isolated by MACS from splenocytes which were
stimulated before in vitro for 2 days with IL-2 (20 ng/ml), IL-2 plus exosomes of Ge-con cells (10 ␮g/ml), or IL-2 plus exosomes of Ge-Hsp70
cells (10 ␮g/ml). b, The mean of CD49b (DX5)-positive NK cells ⫹ SD
among the effector cells used in the three experiments is shown as determined by flow cytometry. c, The inhibition of specific lysis of target cells
which express NKG2D ligands by soluble mouse NKG2D is shown as
determined in an experiment representative for three tests. The means of
specific lysis ⫹ SD of target cells by NK cells at an E:T ratio of 10:1 are
shown. YAC-1, Ge-con, and Ge-con cells exposed to 10 ␮M SAHA for
20 h before the tests served as targets. NK cells were isolated by MACS
from splenocytes which were stimulated before in vitro for 2 days with
IL-2 (20 ng/ml), IL-2 plus exosomes of Ge-con cells (10 ␮g/ml), or IL-2
plus exosomes of Ge-Hsp70 cells (10 ␮g/ml). For inhibition of lysis soluble mouse NKG2D-Fc was added to the test at a concentration of 3 ␮g/ml.
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presence of potentially dangerous cells to the immune system
(43, 49).
Herein we show that HSP70-positive exosomes derived from
HSP70-overexpressing tumor cells activate in vitro mouse NK
cells to kill tumor cells which express NKG2D ligands either constitutively, such as mouse YAC-1 cells, or in an inducible manner,
such as the human Ge melanoma cells. The stimulation of splenocyte cultures by HSP70-positive exosomes increased the capability
of the NK cells to kill targets. This stimulatory effect on NK cells
seems to be a specific property of the stress-inducible HSP70. Exosomes which are released from the HSP70-overexpressing melanoma cells contained the stress-inducible HSP70 in contrast to
exosomes from the HSP70-negative control cells. The cytotoxic
activity of NK cells could also be stimulated by soluble HSP70
(own unpublished results). Thus, HSP70 released from exosomes
might directly contribute to the observed effects.
The cytotoxic activity of NK cells could be inhibited by soluble
NKG2D, confirming the requirement of NKG2D ligands for target
cell recognition by the HSP70-stimulated NK cells. It might be
important that the naive NK cells were stimulated first by the
HSP70-positive exosomes. These preactivated NK cells were subsequently in the effector phase able to kill target cells which expressed NKG2D ligands. Strong and prolonged ectopic expression
of NKG2D ligands such as MICA/B has been reported to result in
an over-stimulation of NKG2D expressing cells, and in a consecutive down-regulation of NKG2D and inactivity of NK cells (50 –
52). Thus, the activation of NK cells might depend on the correct
order and strength of the HSP70 and MICA/B signals.
In the melanoma cell lines used in this study, we observed an
induction of the NKG2D ligands MICA/B in vivo in tumors. In
vitro MICA/B was induced only in response to genotoxic stress,
i.e., the chromatin-modifying treatment with the histone deacetylase inhibitor SAHA (31), but not in response to the proteotoxic
stress of a heat shock, as it has been described for other cell lines
(21, 22, 47). Our findings are in agreement with a recent report
showing that mouse and human NKG2D ligands were up-regulated in several cell lines by various genotoxic stresses but not by
heat shock (48). Therefore, the regulation of NKG2D ligands
might vary between cell lines. It might be interesting that in our
melanoma cells HSP70, which was inducible by proteotoxic stress,
and NKG2D ligands, which were inducible by genotoxic stress,
concordantly promoted the cytotoxic activity of NK cells against
these tumors. Thus, two signals which appear to be independently
5532
Acknowledgments
We thank Prof. E. Günther for his invaluable contributions to initial
phases of this study and Prof. A. G. Pockley for his comments on the
paper. Ralf Dressel thanks Prof. W. Engel and Prof. J. Wienands for
further support. The technical assistance of R. Streich, N. Westphal, and
E. Munk is gratefully acknowledged.
Disclosures
L. Elsner and R. Dressel declare that a patent application has been filled in
describing the use of HSP70 and the HSP70-derived peptide TKD for
activation of NK cells against tumors expressing NKG2D ligands. G. Multoff is in addition to her academic position CEO of the small company
multimmune.
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regulated by different kinds of stress can activate synergistically
the NK cell cytotoxicity.
The activation of NK cells by HSP70 against target cells which
express NKG2D ligands appears to be relevant for immune surveillance of tumors in vivo, in that the growth of HSP70-overexpressing Ge melanoma cells was significantly reduced in SCID
mice. Moreover, in contrast to control tumors the HSP70-overexpressing tumors did not grow invasively and did not give rise to
regional metastases. These effects in SCID mice could unambiguously be attributed to NK cells, because tumor growth and the
rate of metastases of HSP70-overexpressing tumors was the same
as observed for control tumors in SCID/beige mice, which lack
functional NK cells in addition to B and T lymphocytes (36 – 40).
Human MICA/B molecules are known to interact structurally
and functionally with mouse NKG2D (23–25). Therefore, we determined the MICA/B expression in the tumors. The Ge melanoma
cells express MICA/B in vivo, but hardly in vitro. It might be
important that the expression of the NKD2D ligands MICA/B was
endogenously regulated in the tumors in our model so that an overstimulation and consecutive down-regulation of NK cell activity
was avoided (50 –52). The MICA/B expression in tumors appears
to be functionally relevant in SCID mice, because the expression
level of both genes, MICA and MICB, was reduced in HSP70overexpressing tumors grown in SCID compared with SCID/beige
mice. The expression of MICA/B was not regulated epistatically
by HSP70. Therefore, we interpret this reduced MICA/B expression as being an example of “cancer immunoediting” (53). MICA/
B-expressing tumor cells likely become a preferential target for
HSP70-activated NK cells present in SCID mice thereby leading to
a loss of those cells. Splenocytes from SCID mice with HSP70overexpressing tumors were ex vivo able to lyse only those melanoma cells in which NKG2D ligands including MICA/B were
induced by pharmacological means. Although the reduced expression of MICA/B in vivo in SCID mice with HSP70-overexpressing
tumors also indicates a potential immune escape mechanism,
HSP70 overexpression in primary tumors and the subsequent activation of NK cells, which can kill MICA/B expressing tumor
cells, was apparently sufficient to completely suppress invasive
growth and regional metastases. In accordance with others studies
(54) we assume that the activity of NK cells was efficient in our
model at an early time point in progression of the disease before
massive tumors were established. This activity led at least to a
delay of tumor growth. In addition, metastasizing cells were apparently completely eradicated by HSP70-activated NK cells.
The outcome of HSP70 overexpression might depend on the
tumor model that we have analyzed. In our model the extracellular
stimulatory effects of HSP70 on the immune system were apparently more important than intracellular effects of HSP70 which
might increase the tumorigenicity of HSP70-overexpressing cells.
We have shown before that HSP70 overexpression led to reduced
HSC70 expression in the Ge melanoma cells and that HSP70 can
replace functions of HSC70 as a chaperone (19). Importantly, the
effects of HSP70 and HSC70 on the stimulation NK cells appear to
be different (41). The strong overexpression of HSP70 in the GeHsp70 cells might have allowed detecting the NK cell stimulatory
effects of HSP70 in our tumor model. However, the endogenous
HSP70 was also induced in vivo in the tumors as shown for
HSP70-2 at the mRNA level. Therefore, HSP70 and NKG2D ligands such as MICA/B might become expressed also in other tumors and contribute together to the activation of NK cells.
HSP70 ACTIVATES MOUSE NK CELLS AGAINST TUMORS
The Journal of Immunology
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
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