The Journal of Immunology
Heat Shock Protein 70, Released from Heat-Stressed Tumor
Cells, Initiates Antitumor Immunity by Inducing Tumor Cell
Chemokine Production and Activating Dendritic Cells via
TLR4 Pathway1
Taoyong Chen,2 Jun Guo,2,3 Chaofeng Han, Mingjin Yang, and Xuetao Cao4
Extracellular heat shock proteins (HSP) can activate dendritic cells (DC) and monocytes/macrophages, and HSP derived from
tumor cells have been regarded as potent adjuvant facilitating presentation of tumor Ags and induction of antitumor immunity.
However, the roles and the underlying mechanisms of releasable HSP in the induction of antitumor immunity have not been fully
elucidated. In this study, we report that heat stress can induce the release of various HSP from tumor cells, which, in turn, activate
tumor cells to produce chemokines for chemoattraction of DC and T cells via TLR4 signaling pathway. In vivo, we find that the
infiltration and function of DC and T cells within tumor after local hyperthermia are increased significantly. We also provide
evidence that HSP70 proteins released by tumor cells and TLR4 expressed by tumor cells/DC are essential for the chemoattraction
of DC/T cells and for the subsequent induction of tumor-specific antitumor immunity. Therefore, our study suggests that heat
stress-induced releasable HSP70 proteins from tumor cells play important roles in the initiation of antitumor immunity by
inducing tumor cell production of chemokines and by activating the chemoattracted DC via TLR4 pathway. The Journal of
Immunology, 2009, 182: 1449 –1459.
T
raditionally, heat shock proteins (HSP)5 are regarded as
chaperones assisting protein folding and translocation.
However, HSP can also serve as cytokines that can stimulate dendritic cells (DC) and macrophages to produce proinflammatory cytokines and chemokines (1–5). More importantly, HSP
derived from tumor cells are capable of chaperoning tumor Ags to
DC and then cross-presenting the Ags to T cells (6). HSP70 proteins, including the constitutively expressed cognate HSP70
(HSC70 or HSP73), the stress-inducible HSP70 (HSP70i or
HSP72), and the mitochondrial HSP70 (HSP75), constitute the
most conserved class of all HSP. Previous reports have shown that
HSP can be released from various cells via passive (e.g., HSP
National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, People’s Republic of China
Received for publication July 10, 2008. Accepted for publication November 24, 2008.
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 from Foundation for the Author of National
Excellent Doctoral Dissertation of China (200775), National Natural Science Foundation of China (30572122, 30771118, and 30721091), National Key Basic Research
Program of China (2007CB512403), and Shanghai Committee of Science and Technology (07QA14067).
2
T.C. and J.G. contributed equally to this work.
3
Current address: Department of Renal Cancer and Melanoma, Beijing Cancer Hospital and Institute, Beijing, People’s Republic of China.
4
released during cell injury conditions, such as surgery, excessive
exercise, and necrosis) and active (e.g., translocation of HSP to
plasma membrane and subsequent secretion) pathways (3–5).
However, the roles of HSP70 proteins released from tumor cells in
the induction of antitumor immunity and the underlying mechanisms have not been fully elucidated.
Hyperthermia (HT) has been reported to enhance the immunogenecity of cancer cells concomitantly with expression of
HSP (7, 8). Recent reports demonstrate that heat stress (HS) can
induce the cell surface expression and the release of HSP70,
HSP90, and gp96 (glucose-regulated protein 94; Grp94) (1–5).
However, the underlying mechanisms that local HT can initiate
antitumor immunity via released HSP and via subsequent activation of DC still lack direct evidence and thus need to be
further investigated.
During the investigations of local HT (42– 43°C)-elicited antitumor immunity, we find that infiltration of DC and T cells
within heat-stressed tumor is significantly increased. We thus
hypothesize that chemokines, induced by HS, may be involved
in the initiation of HT-elicited antitumor immunity by chemoattraction and activation of DC. Our studies show that HSP70
proteins simultaneously released by tumor cells can serve as
autocrine and paracrine cytokines inducing the production of
various chemokines by tumor cells and the activation of DC via
TLR4 signaling pathway. Our data provide direct evidence for
the important roles of releasable HSP in the initiation of antitumor immunity during local HT.
Address correspondence and reprint requests to Dr. Xuetao Cao, National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China.
E-mail address: [email protected]
Materials and Methods
5
Mice, cells, Abs, and reagents
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
Male wild-type C57BL/6 (H-2Kb) mice, 6 – 8 wk of age, were obtained
from SIPPR-BK Experimental Animal Company. TLR4 knockout mice
(TLR4⫺/⫺,C57BL/6 strain) were provided by Dr. S. Akira (Research Institute for Microbial Diseases, Osaka, Japan) (9). All mice were housed in
a specific pathogen-free facility for all experiments. Murine Lewis lung
carcinoma 3LL and murine malignant melanoma B16 cells were obtained
Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cell; DC,
dendritic cell; HS, heat stress; HSC70, cognate HSP70; HSP70i, inducible HSP70;
HSP, heat shock protein; HT, hyperthermia; MDSC, myeloid-derived suppressor cell;
RNAi, RNA interference; siRNA, small interfering RNA; SN, culture supernatant;
TIMC, tumor-infiltrating mononuclear cell; Treg, regulatory T cell; WCL, whole-cell
lysate; TRIF, Toll/IL-1R domain-containing adapter inducing IFN␤.
www.jimmunol.org
1450
RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4
from American Type Culture Collection. These cell lines were maintained
in appropriate medium as recommended. Mouse bone-marrow derived DC
(BMDC) were prepared by culturing with 20 ng/ml rGM-CSF and 10
ng/ml IL-4 (Genzyme) as described previously (10). The mAbs against
HSP70i, HSC70, HSP60, HSP90 (recognizing both HSP90␣ and HSP90␤),
gp96, CD91, CD14, TLR2, and TLR4, as well as the rHSP70i, HSC70,
HSP90, and HSP60 proteins, were obtained from Abcam. Abs specific for
total and phosphorylated forms of IFN regulatory factor 3 (IRF3; Ser396)
and I␬B␣ (Ser32/36) were obtained from Cell Signaling Technology. Fluorescent Abs against CD80, CD86, Iab, CD40, CD11c, CD3, CD4, CD8,
CD25, DX5, Gr1, FoxP-3, and isotype control Abs were obtained from BD
Pharmingen. The rHSP proteins were further deprived of LPS contamination as described by using polymycin B agarose (Sigma-Aldrich) (11). The
purity (⬎92%) and LPS contamination (⬍5 pg/␮g protein) of rHSP were
determined as described previously (12). ELISA kits for measuring chemokines and cytokines were purchased from R&D Systems. Other nonspecified reagents were purchased from Sigma-Aldrich.
Establishment and monitor of tumor model, local HT treatments,
and immunological assessment of systemic antitumor immunity
3LL cells (5 ⫻ 105) in 100 ␮l of PBS were s.c. injected into the shaved
right flanks of the C57BL/6 mice (H-2Kb). Seven days after the inoculation, the tumors (100 –150 mm3) were treated by using a 915-MHz
microwave machine for superficial tumor at 42– 43°C for 1 h at an
interval of 1 wk for a total of three times. Body surface temperature was
maintained at 42– 43°C, which was confirmed by a thermosensor during
HT for superficial tumors as previously described (13), and could induce significant increase of HSP expression as determined by Western
blot assays. Tumor sizes were measured every 2–3 days, and the tumor
volumes were determined by measuring of the maximal (a) and minimal
(b) diameters using a caliber and calculated by using the formula a ⫻
b2. The survival of the tumor-bearing mice was observed daily as described previously (14). Mice were sacrificed when the tumors reached
2 cm in diameter or appeared moribund, and this was recorded as the
date of death for survival studies.
For assessment of systemic CTL induction, 12 days after the last
treatment of tumor with HT, CTL activity was measured by using a
standard 4-h 51Cr release assay as described previously (14). 3LL cells
were used as targets and syngeneic lymphoma EL-4 cells were used as
control targets.
Immunohistochemistry
Different hours after the first treatment of HT, tumors were isolated, frozen
sectioned, and examined for the infiltrations of DC and T cells as described
previously (14). The number of immunostained cells was examined by
light microscopy.
Isolation of tumor-infiltrating mononuclear cells, DC, and
T cells
For the isolation of tumor-infiltrating mononuclear cells (TIMC), tumor
tissues were cut into small fragments and incubated in RPMI 1640 medium
containing 1 mg/ml collagenase (4 ml/g tissue) and 0.25% DNase I at 37°C
for 45 min as described previously (10). Then, single-cell suspensions of
TIMC, lymph node mononuclear cells, and splenocytes were prepared and
collected by centrifugation on a Ficoll gradient. For the isolation of DC and
T cell populations, the isolated immune cell suspensions were incubated
with magnetic beads specifically for CD11c⫹, CD3⫹, CD4⫹, or CD8⫹
markers and then isolated by immunomagnetic separation (MACS; Miltenyi Biotec) on RS1 columns as described previously (10, 13).
HS treatments of tumor cell lines
Tumor cells (3LL and B16) growing in 60-ml flasks (4⬃6 ⫻ 106 cells
in 5 ml medium) were heat treated at 42°C in an air incubator containing 5% CO2.
upon request. Quantitative PCR was performed on a MJR Chromo4 Continuous Fluorescence detector (Bio-Rad) according to the manufacturer’s
protocol and as described previously (15).
Western blotting
Total cell lysates were prepared as described previously, and protein concentration was determined by the bicinchoninic acid protein assay (Pierce).
Cell extracts were subjected to SDS-PAGE, transferred onto nitrocellulose
membrane, and blotted as described previously (10).
RNA interference (RNAi)
For transient silencing of HSP, 21-nt sequences of small interfering RNA
(siRNA) duplexes were synthesized as follows: 5⬘-GGU GGA GAU CAU
CGC CAA CUU-3⬘ (sense) and 5⬘-GUU GGC GAU GAU CUC CAC
CUU-3⬘ (antisense) for HSP70i; 5⬘-UGA ACC CCA CCA ACA CAG
UUU-3⬘ (sense) and 5⬘-ACU GUG UUG GUG GGG UUC AUU-3⬘ (antisense) for HSC70; 5⬘-ACC CAG ACC CAA GAC CAA CUU-3⬘ (sense)
and 5⬘-GUU GGU CUU GGG UCU GGG UUU-3⬘ (antisense) for
HSP90␣; 5⬘-GUG CAC CAU GGA GAG GAG GUU-3⬘ (sense) and 5⬘CCU CCU CUC CAU GGU GCA CUU-3⬘ (antisense) for HSP90␤; 5⬘GAA GCU AUU CAG UUG GAU GUU-3⬘ (sense) and 5⬘-CAU CCA
ACU GAA UAG CUU CUU-3⬘ (antisense) for gp96; and 5⬘-UGC UUC
AAG GUG UAG ACC UUU-3⬘ (sense) and 5⬘-AGG UCU ACA CCU
UGA AGC AUU-3⬘ (antisense) for HSP60. The corresponding sequences
containing two nucleotide mutations were used as a scrambled control
(Ctrl). All the used sequences were blasted against the National Center for
Biotechnology Information nucleotide database to exclude nonspecific
interference. siRNA duplexes were transfected into tumor cells using
INTERFERin reagent (Polyplus-transfection) according to the standard
protocol and as described previously (15). For exclusion of the possible
off-targets effects of HSP70i siRNAs, HSP70i sequence containing two
synonymous mutations (G84 to C and C96 to G, ␮-HSP70i) in pcDNA3.1
vector (Invitrogen) was transfected into 3LL cells.
For stable knockdown of TLR4, CD91, CD40, and CD14 expression in
3LL tumor cells, an expression vector (psilencer-U6 neo; Ambion) with
insertion of specific siRNA duplexes of the indicated receptors or the corresponding scrambled siRNA duplexes were transfected into 3LL cells
using Jetpei (Polyplus-transfection) according to the standard protocol and
as described previously (15). The targeting 19-nt sequences used were as
follows: 5⬘-GCT TGA ATC CCT GCA TAG A-3⬘ for TLR4, 5⬘-CTT CTC
AGA TCC GAA GCC A-3⬘ for CD14, 5⬘-AAC TTG CAG CCC TAA
GCA G-3⬘ for CD91, and 5⬘-GCC GAC TGA CAA GCC ACT G-3⬘ for
CD40. For stable silence of both HSP70i and HSC70, we used the sequences targeting 5⬘-TCC TAT GCC TTC AAC ATG A-3⬘ for construction of RNAi vectors, which was less efficient than that used for transient
silence and could lead to ⬃70% down-regulation of both HSP70i and
HSC70 proteins. After transfection of the RNAi vectors, cells were selected with 500 – 800 ng/ml neomycin for 2 wk.
Assay of NF-␬B and IRF3 activation
For analysis of NF-␬B and IRF3 signaling pathways, phospho-Abs against
I␬B␣ and IRF3 were used to detect the expression levels of these molecules in whole-cell lysates (WCL) by Western blot. To examine the nuclear
translocation of NF-␬B and IRF3, the extracted nuclear proteins, using
NE-PER nuclear reagents (Pierce), were Western blotted to detect the presence of NF-␬B p65 subunit and IRF3 in the nucleus as described previously (10, 15).
Luciferase reporter assay
The determination of NF-␬B and IRF3 transactivation was performed as
described previously (15). The pGL3.5X␬B-luciferase reporter plasmid
was provided by S. J. Martin (Trinity College, Dublin, Ireland) (16). The
IRF3 reporter plasmids were gifts from Dr. T. Fujita (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan) (17). The pRL-TK-Renillaluciferase plasmid was obtained from Promega.
In vitro chemoattraction assay
To evaluate the chemotactic activity of culture supernatants (SN) from
tumor cells, chemotaxis of mouse DC and splenic T cells was assayed as
described previously by us (10).
RT-PCR and quantitative PCR
Total cellular RNA was extracted using TRIzol reagent (Invitrogen). RTPCR was performed as described previously (10). Specific primers used for
RT-PCR assays of chemokines, chemokine receptors (CXCR4 and CCR7),
and HSP receptors (TLR4, TLR2, CD40, CD14, and CD91) were available
Functional assessment of DC
To evaluate the functional status of DC isolated from tumor tissues (enriched from TIMC with CD11c-specific magnetic beads), DC were subjected to the assays of phenotype, cytokine secretion assay, and allogeneic
stimulatory capacity assay as described previously (10).
Functional assessment of tumor-infiltrating T cells
To evaluate the functional status of tumor-infiltrating T cells, CD3⫹ cells
isolated from TIMC using immunomagnetic beads were cultured in vitro
The Journal of Immunology
1451
FIGURE 1. Local hyperthermia
promotes the infiltration of DC and T
cells in vivo. A, FACS assays of infiltrated DC (CD11c⫹), T cells
(CD3⫹CD4⫹ or CD3⫹CD8⫹) and
NK cells (DX5⫹) within the TIMC
populations of 3LL tumors treated
with (3LL HT) or without (3LL) HT
for 1 h and recovered for 8 h. The
results were representative images
obtained from five mice, and the
numbers indicated for percentages of
cells to all the TIMC populations. B,
FACS analysis of Treg and MDSC
populations. The isolated TIMC cells
were purified for CD4⫹ and CD11b⫹
populations and then stained for
FoxP-3/CD25 and Gr1 as indicated.
Numbers indicated for percentages
CD25⫹FoxP-3⫹ (upper panel) and
Gr1⫹ (lower panel) cells to all the
CD4⫹ and CD11b⫹ cells respectively. C, FACS assays of DC
(CD11b⫹ cells), NK (DX5⫹ cells),
Treg (CD4⫹CD25⫹Fox-P3⫹ cells),
and MDSC (Gr1⫹CD11b⫹ cells)
within TIMC derived from 3LL tumors treated with (1-h HT plus 4-, 8-,
12-, and 24-h recovery, respectively)
or without (0 h) HT. Results were
presented as percentages of indicated
cells to total TIMC and expressed as
mean ⫾ SEM of five mice. ⴱ, p ⬍
0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.
and stimulated with 2 ␮g/ml anti-CD3 and anti-CD28 mAb (BD Pharmingen) and then examined for the proliferation and cytokine production
capacity as described previously (18, 19).
In vitro CTL induction and cytotoxicity assays
To evaluate the DC-T cell interaction within spleen after HT treatments in
vitro, enriched CD11c⫹ (2 ⫻ 105 DC/well) and CD8⫹ cells derived from
spleen of HT-treated mice were cocultured in vitro in medium containing
30 U/ml IL-2 (PeproTech) for 7 days in 24-well plates in a final volume of
2 ml/well (1 DC per 10 CD8⫹ T cells). Every 2 days, 0.5 ml of supernatant
was replaced with fresh medium. Seven days later, functions of stimulated
CD8⫹ T cells were evaluated by IFN-␥ release assay and cytotoxicity assay
as described previously (18, 19). To examine 3LL-specific CTL induction,
we prepared BMDC from normal wild-type mice after in vitro cultured for
5 days, matured them with 100 ng/ml LPS stimulation for 48 h, pulsed them
with 50 ␮g/ml synthetic MHC-I-restricted peptides MUT1 (FEYNYAQL,
high affinity for H-2Kb) and OVA (257–264, SIINFEKL, H-2Kb) at the
final 1 h (20, 21), and irradiated (5000 rad) them. For IFN-␥ release assay,
stimulator cells (DC pulsed with MUT1) were cocultured with the stimulated CD8⫹ T cells at different ratios. Forty-eight hours later, the SN were
tested for the level of IFN-␥ by ELISA. Cytotoxicity assays were performed using standard 4-h 51Cr release assay against labeled target cells as
described (14, 19). Targets for CTL activity were mature DC pulsed with
MUT1 or OVA peptides for 1 h.
Statistical analysis
All experiments were independently performed three times. Results are
given as means ⫾ SE or means ⫾ SD. Comparisons between two groups
were done using Student’s t test, while comparisons between multiple
groups were done using Kruskal-Wallis tests. Survival estimates and median survivals were determined using the method of Kaplan and Meier.
Statistical significance was determined as values of p ⬍ 0.05.
Results
Local HT increases the infiltration of DC and T cells but
relatively decreases the infiltration of regulatory T cells (Treg)
and myeloid-derived suppressor cells (MDSC) in the tumor
tissue
After treatments of pre-established 3LL tumor with HT, we found
that HT could inhibit the in vivo growth of 3LL Lewis lung carcinoma (Supplemental Fig. S1A)6 and prolong the survival of the
tumor-bearing mice (Supplemental Fig. S1B). CTL assays indicated that HT could elicit potent 3LL tumor-specific antitumor
immunity (Supplemental Fig. S1C).
We then investigated the infiltration of immune cell populations
within the tumor tissue by immunohistochemistry. We found that
the tumor infiltrations of DC (CD11c⫹) and T cells (CD4⫹ or
CD8⫹) at different time points after the first treatment of HT were
increased significantly (Supplemental Fig. S2). To more accurately
assess the infiltration of DC and T cells within tumor tissue after
HT, we isolated TIMC 8 h after the first HT treatment and analyzed the cell populations within tumor by FACS. We found that
the numbers of DC (CD11c⫹), T cells (CD3⫹CD4⫹ and CD3⫹
CD8⫹), and NK cells (NK, DX5⫹) were significantly increased
after HT (Fig. 1A). We further examined Treg (CD4⫹CD25⫹
FoxP3⫹) and MDSC (Gr1⫹CD11b⫹) within tumor after HT. We
found that the percentages of Treg and MDSC cells in TIMC were
6
The online version of this article contains supplemental material.
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RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4
FIGURE 2. Local HT improves the functional status of infiltrated DC and T cells within tumor. TIMC populations were isolated from tumor tissue before
HT (3LL) or 24 h after the 1-h HT treatments (3LL HT), and then DC and T cells were enriched using magnetic beads specific for CD11c and CD3,
respectively. A, FACS assays of DC phenotype. The enriched DC were stained with fluorescent Abs against Iab, CD80, CD86, and CD40 as indicated.
Numbers indicated for mean fluorescence intensity of the representative results. B, Cytokine production of DC. The enriched DC in 24-well plates (1 ⫻
105/ml) were stimulated with (24 h) or without (0 h) LPS (1 ␮g/ml) as indicated, and then the supernatants were examined for IL-12p70 and TNF-␣
production by ELISA. Results were presented as mean ⫾ SD of triplicate samples. C, MLR assay of DC. The enriched DC were treated with 1 ␮g/ml LPS
or medium alone for 48 h, irradiated as stimulator cells, and then cocultured with allogeneic splenic T cells (BALB/c) at different responder:stimulator ratios.
Results were presented as mean ⫾ SD of triplicate samples. D, CXCR4 and CCR7 expression by DC. The enriched DC were treated with or without (0
h) 1 ␮g/ml LPS for 24 h (for quantitative RT-PCR assays) or 48 h (for Western blot assays) as indicated. For quantitative RT-PCR assays (left two panels),
results were expressed as fold induction of mRNA to that of DC derived from TIMC of mice without HT treatments and presented as mean ⫾ SD of
triplicate samples. E and F, Cell proliferation (E) and cytokine production (F) assays of T cells. The enriched T cells were treated with 2 ␮g/ml anti-CD3
and anti-CD28 (anti-CD3/CD28) Ab as indicated. Then, proliferation was examined by [3H]thymidine incorporation assays (E), and cytokines in the
supernatants were examined by ELISA (F). Results were presented as mean ⫾ SD of triplicate samples. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.
decreased (Fig. 1B). We then examined the infiltrating DC
(CD11c⫹), NK (DX5⫹), Treg (CD4⫹CD25⫹FoxP3⫹), and MDSC
(CD11b⫹Gr1⫹) within TIMC at different time points after the first
HT, and we found that the infiltration of these populations of cells
was increased in total numbers after HT (data not shown), and the
percentages of immunosuppressive cell populations (MDSC and
Treg) of TIMC were relatively decreased due to the increase of
infiltrated cells (Fig. 1C), which may finally favor the observed
inhibition of tumor growth and induction of tumor-specific CTL in
vivo (Supplemental Fig. S1). As further evidence, we treated B16
melanoma-bearing tumor models with local HT and found that HT
could inhibit tumor growth, improve survival and promote the infiltration of DC and T cells at a similar extent to that observed in
3LL tumor models (data not shown).
Local HT improves local and systemic activation of DC and
T cells
We then isolated the tumor-infiltrating DC and T cells from TIMC
by magnetic beads specific for CD11c⫹ and CD3⫹ cells, respectively, with purity ⬎90%. We found that DC-derived from the
The Journal of Immunology
1453
HT-treated tumor (8 h after the first HT treatment) showed elevated expression levels of surface Iab, CD80, CD86, and CD40
(Fig. 2A). Moreover, such DC showed more potent capacity in
producing IL-12p70 and TNF-␣ (Fig. 2B) and in stimulating the
proliferation of allogeneic CD3⫹ T cells (Fig. 2C). Also, the
expression of CCR7 and CXCR4 were significantly increased in
such DC (Fig. 2D). These data suggested that HT treatment not
only increased the numbers of infiltrated DC (Fig. 1) but also
improved the maturation and Ag presentation capacity of these
tumor-infiltrating DC.
To examine the functional status of tumor-infiltrating T cells, we
incubated the enriched CD3⫹ T cells with anti-CD3/CD28 Ab for
48 h and then found that T cells derived from HT-treated tumors
showed elevated capacity in proliferation (Fig. 2E) and increased
production of IL-2 and IFN-␥ while decreased production of
TGF-␤ and IL-10 (Fig. 2F). These data suggested that the functional status of T cells within tumors after HT was also improved.
Then, we examined the migration of DC and T cells in the
spleen and draining lymph nodes of tumor-bearing mice after local
HT. We found that local HT could significantly increase the percentage of DC within these secondary lymphoid organs (Fig. 3A),
suggesting that HT might promote DC to migrate to spleen and LN
for tumor Ag presentation. To test this possibility, we isolated
splenic CD11c⫹ DC from 3LL tumor-bearing mice with or without HT treatments, cocultured them with splenic CD8⫹ T cells
derived from 3LL tumor-bearing mice, and examined the CTL
induction. We found that splenic DC derived from HT-treated 3LL
tumor-bearing mice could induce more IFN-␥ production (Fig. 3B)
and more potent MUT1-specific cytotoxicity of CD8⫹ T cells (Fig.
3C). These data suggest that local HT may promote activation of
DC and CD8⫹ T cells in the secondary lymphoid organs.
Heat-stressed tumor cells release chemokines and subsequently
chemoattract DC and T cells in vitro
Chemokines play essential roles in the regulation of DC residency
in peripheral tissues and DC migration to secondary lymphoid organs (22–24). Therefore, we hypothesized that local HT could promote the production of chemokines by tumor cells. We found that
the SN derived from HS-treated 3LL tumor cells could chemoattract more CD11c⫹ DC and CD3⫹ T cells than that from untreated
3LL cells (Fig. 4A). Similar results were observed when B16 melanoma and other tumor cell lines were used as alternative models
(data not shown).
To examine whether HS could induce chemokine production in
3LL cells, we performed RT-PCR assays of chemokines in 3LL
tumor cells before and after HS treatments and found that HS
treatments could significantly increase the expression of chemokines, including CC chemokines (CCL2/MCP-1, CCL3/MIP-1␣,
CCL4/MIP-1␤, CCL5/RANTES, CCL19/MIP-3␤, CCL20/MIP3␣, and CCL25/thymus-expressed chemokine), CXC chemokine
(IP-10), and CX3C chemokine (CX3CL1/fracktalkine) (Supplemental Fig. S3). We examined the tumor cell production of CCL2,
CCL5, and CXCL10 by both quantitative RT-PCR and ELISA and
confirmed that these chemokines were all significantly increased
by HS treatments (Fig. 4, B and C).
Releasable HSP70 derived from tumor cells after HS are
required for the chemoattraction of DC
Up-regulation of HSP is a typical effect of HS on cells, and
releasable HSPs have been shown to function as cytokines to
activate DC and macrophages (3, 4). We then hypothesized that
HS may induce chemokine expression of tumor cells by the
released HSP.
FIGURE 3. Local hyperthermia promotes the presence of DC within
spleen and lymph nodes and the induction of CTL. A, Percentages of
CD11c⫹ cells within spleen and lymph node. The 3LL tumor-bearing mice
were treated for 1 h by local HT (3LL HT), and the spleen and lymph nodes
of the mice were isolated for mononuclear cells 24 h after HT. Then, DC
(CD11c⫹) in the mononuclear cells were analyzed by FACS assays, and
the results were representative data derived from five mice and presented
as percentages of CD11c⫹ cells within all the mononuclear cells. B and C,
In vitro CTL induction assays. Forty-eight hours after HT treatments,
CD11c⫹ cells were enriched from the spleen by magnetic beads. The
splenic CD11c⫹ cells derived from HT-treated mice (3LL HT) or nontreated mice (3LL) were cocultured with splenic CD8⫹ T cells derived
from spleens of nontreated 3LL tumor-bearing mice for 7 days at a ratio of
1:10. To trigger IFN-␥ release (B), we used the in vitro-cultured BMDC
(on day 5) matured with 100 ng/ml LPS for 48 h and pulsed with 10 ␮g/ml
high-affinity MUT1 peptides at the last 1 h as stimulators. C, The matured
BMDC (100 ng/ml LPS for 48 h) pulsed with MUT1 peptides (MUT1-DC)
or control OVA (257–264) peptides (OVA-DC) were used as targets at
indicated ratios. The MUT1-specific cytotoxicity was examined by 51Cr
release assay. Results were presented as mean ⫾ SD of triplicate samples.
ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
To test this possibility, we first examined the release of HSP
by 3LL cells after HS treatments. We found that HSP90, gp96,
HSC70, HSP70i, and HSP60 were all rapidly released into extracellular medium after HS treatments within 10 min (Fig. 5A).
During the 1 h of HS treatment, HSP90, gp96, HSP70i, and
HSP60 in the cell lysates were not significantly elevated at protein level (data not shown), indicating that HS induced the active release of HSP. We then blocked the released HSP by using
1454
RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4
FIGURE 4. SN from heat-stressed
tumor cells contain chemokines and
can chemoattract DC and T cells in
vitro. A, In vitro chemoattraction assays of the SN derived from nontreated (3LL SN) or heat-treated (3LL
HT SN) cells toward DC (left panel)
and T cells (right panel). DC were in
vitro-cultured immature BMDC (day
5), and T cells were splenocytes derived from wild-type C57BL/6 mice.
Medium was used as negative control. Results were presented as
mean ⫾ SD of triplicate samples. ⴱⴱ,
p ⬍ 0.01. B and C, HS induces chemokine production of 3LL cells. 3LL
cells (0 h) were subjected to HS treatments at 42°C for 1 h and then recovered at 37°C for 4 and 8 h as indicated. Then, 3LL cells were used for
quantitative RT-PCR assays of chemokine production (B), and the SN
were measured for chemokine production by ELISA (C). Results were
presented as mean ⫾ SD of triplicate
samples. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01;
ⴱⴱⴱ, p ⬍ 0.001.
neutralizing Abs and performed the in vitro chemoattraction
assays. We found that blocking Abs specific for HSC70 and
HSP70i could significantly inhibit the capacity of HS-treated
3LL SN to chemoattract DC (Supplemental Fig. S4A). And to a
lesser extent, blocking Abs for HSP90 and gp96, but not
HSP60, could also inhibit the chemotactic activity of the HStreated 3LL SN (Supplemental Fig. S4A). Similarly, supplement
of rHSP into the SN of 3LL without HS treatments could elicit
similar chemotactic effects on DC (Supplemental Fig. S4B),
whereas rHSP alone in the medium could not chemoattract DC
(data not shown). These data suggested that the released HSP
(mainly HSP70) derived from 3LL cells after HS were responsible for the observed chemotactic activity of HS-treated 3LL
SN toward DC.
To investigate which kind of HSP plays prominent roles in eliciting chemotactic activity via inducing chemokine production, we
silenced the expression of HSP60, HSP70s, HSP90s, and gp96 via
transient transfection of siRNA (Supplemental Fig. S5). We found
that the chemotactic activity of SN derived from HSP70 (both
HSC70 and HSP70i)-silenced cells after HS treatment toward DC
decreased significantly (Fig. 5B). Moreover, similar to those observed in Ab blocking experiments (Supplemental Fig. S4A),
HSP90 and gp96 silence could also reduce the HS-treated 3LL
SN-mediated chemoattraction of DC (to a much lesser extent),
whereas HSP60 silence has no significant effects on the chemotactic activity toward DC (Fig. 5B).
Releasable HSP70 derived from tumor cells after HS
up-regulates the expression of chemokines in tumor cells via
TLR4-mediated signaling pathway
We next examined the roles of HSP70 in the induction of chemokine production by tumor cells during HS treatments. We found
that the HS-induced up-regulation of the chemokines’ mRNA
was significantly reduced in HSP70 (both HSC70 and HSP70i)silenced 3LL cells, as compared with that in scrambled control
siRNA-silenced 3LL cells (Ctrl RNAi; Fig. 5, C and D). To
exclude the possibility that the above decrease of chemotactic
activity by HSP70 silence was due to the roles of intracellular
HSP70 during HS treatments, we supplemented rHSP70
(HSC70 ⫹ HSP70i) in the SN of HSP70-silenced 3LL cells
during HS treatments. We found that the supplement of rHSP70
proteins could rescue the observed reduction of DC chemoattraction (Fig. 5B) and chemokine production by HSP70 silence
(Fig. 5, C and D). For exclusion of possible off-targets of
HSP70 siRNA, we transiently transfected the HSP70-silenced
3LL cells with ␮-HSP70i vectors (resistant to HSP70 siRNA;
Fig. 5E). We found that the overexpression of ␮-HSP70i could
rescue the inhibitory effects of HSP70 siRNA on HS-induced
chemoattraction of DC and CXCL10 production (Fig. 5, F and
G). These results suggested that the released HSP70 (including
HSP70i and HSC70) in the SN of HS-treated 3LL cells played
essential roles in the chemoattraction of DC and T cells by
inducing tumor cell production of chemokines via an autocrine
manner.
Releasable HSP can activate target cells via receptor binding
and signaling (25–34). We thus examined the expression of TLR2,
TLR4, CD40, CD91, and CD14 in 3LL tumor cells. We found that
TLR4, CD40, CD91, and CD14, but not TLR2, were all expressed
by 3LL cells to a different extent, and HS treatments could elevate
the expression of these receptors (Supplemental Fig. S6A). Then,
we wondered which receptor(s) was responsible for the induction
of chemokines by HSP70 during HS treatments. We thus stably
silenced the expression of CD91, CD14, CD40, and TLR4 in 3LL
cells (Supplemental Fig. S6B) and then examined the induction of
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1455
FIGURE 5. HSP70 silence inhibits tumor
cell chemokine production and subsequent chemoattraction of DC induced by HS. A, HS rapidly induces the release of HSP into medium.
3LL tumor cells were subjected to HS treatments (42°C) as indicated. Then, SN were collected by centrifugation at 13,000 ⫻ g for 10
min. HSP contained in the SN were examined by
Western blot as indicated. BSA supplemented in
the SN were probed as quantitative control. B,
HSP silence affects the chemoattraction of SN
toward BMDC. 3LL cells silenced with siRNA
targeting HSP90, gp96, HSP70 (both HSC70
and HSP70i), and HSP60 for 48 h were HS
treated for 1 h and recovered for 8 h. Then, SN
were collected and subjected to in vitro chemoattraction assays. Results were expressed as
number of CD11c⫹ cells that have migrated to
lower chamber as counted by FACS and presented as mean ⫾ SD of triplicate samples. Œ,
p ⬎ 0.05; ⴱⴱⴱ, p ⬍ 0.001. C and D, HSP70
silence inhibits HS-induced chemokine production. 3LL cells silenced with scrambled siRNA
duplexes (ctrl siRNA) or HSC70/HSP70i
siRNA (HSP70 siRNA) for 48 h were treated
with HS for 1 h plus 4-h recovery (for quantitative RT-PCR) or 8-h recovery (for ELISA)
or treated without HS (non-HS). Then, cells
and SN were analyzed for mRNA (C) and protein (D) expression of CXCL10, respectively.
Results were presented as mean ⫾ SD of triplicate samples. Œ, p ⬎ 0.05; ⴱⴱⴱ, p ⬍ 0.001.
E, ␮-HSP70i overexpression in HSP70-silenced cells. 3LL cells silenced with scrambled siRNA duplexes (ctrl siRNA) or HSC70/
HSP70i siRNA (HSP70 siRNA) were
transiently transfected with mock vector or
␮-HSP70i vector containing synonymous mutations resistant to HSP70 siRNAs and encoding HSP70i. WCL and SN treated with HS for
1 h were examined by Western blot. F and G,
␮-HSP70i overexpression attenuates HSP70
silence-induced inhibition of DC chemoattraction (F) and chemokine production (G) after
HS (1-h HS plus 8-h recovery). Œ, p ⬎ 0.05;
ⴱⴱⴱ, p ⬍ 0.001.
chemokines in these silenced 3LL cell by HS treatments. We found
that TLR4-silenced 3LL cells showed significantly decreased
production of CXCL10, whereas the silence of CD14 also could reduce the production of CXCL10 to some extent (Fig. 6, A and
B). However, the silence of CD91 and CD40 did not affect the
production of these chemokines (data not shown). Moreover,
we found that TLR4 silence didn’t affect the HS-induced release
of HSP70 and HSC70 by tumor cells (data not shown). However, the SN derived from TLR4-silenced, HS-treated 3LL cells
showed significantly decreased chemotactic activity toward DC
(Fig. 6C).
As further evidence, we examined the TLR4 signaling in TLR4silenced 3LL cells after HS treatment. We found that the phosphorylation of I␬B␣ and the nuclear translocation of NF-␬B p65
subunit were significantly reduced in TLR4-silenced 3LL cells as
compared with that in scrambled control siRNA-silenced 3LL cells
after HS treatment (Fig. 6D). Moreover, the phosphorylation and
nuclear translocation of IRF3 were also significantly reduced in
TLR4-silenced 3LL cells (Fig. 6D). In addition, we found that both
NF-␬B and IRF3 gene reporter activation decreased significantly
by TLR4 silence during HS treatments of tumor cells (Fig. 6E).
These data suggested that HS-induced activation of both
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RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4
FIGURE 6. TLR4 silence inhibits tumor cell chemokine production and subsequent chemoattraction of DC induced by HS. A and B, Chemokine
production of TLR4- or CD14-silenced 3LL cells. 3LL cells stably silenced with TLR4 silencing vector (TLR4 RNAi; A), CD14 silencing vector
(CD14 RNAi; B), or scrambled control silencing vector (Ctrl RNAi) were treated with HS for 1 h plus 4-h recovery (for quantitative RT-PCR) or
8-h recovery (for ELISA) or treated without HS (non-HS). Then, cells and SN were analyzed for mRNA (left panels) and protein (right panels)
expression of CXCL10, respectively. Results were presented as mean ⫾ SD of triplicate samples. ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001. C, In vitro
chemoattraction of DC. Indicated 3LL cells were treated with HS (1 h plus 8-h recovery), and those cells not treated with HS (non-HS) were collected
for SN. The chemoattraction of DC was expressed as number of CD11c⫹ cells that have migrated to lower chamber as counted by FACS and
presented as mean ⫾ SD of triplicate samples. ⴱⴱⴱ, p ⬍ 0.001. D, Western blot assays of NF-␬B and IRF3 activation. The indicated cells were treated
with HS as indicated. Then, WCL and nuclear proteins (nucleus) were prepared and blotted for phosphorylated I␬B␣ (p-I␬B␣) and IRF3 (p-IRF3),
total IRF3 (t-IRF3) and p65 subunit of NF-␬B. ␤-Actin and lamin A were examined as loading control. E, NF-␬B and IRF3 reporter assay. 3LL cells
stably silenced with TLR4 silencing vector (TLR4 RNAi) or scrambled control vector (Ctrl RNAi) were transiently transfected with luciferase
reporter plasmids of NF-␬B or IRF3 and pTK-Renilla-luciferase vectors. Forty-eight hours later, cells were treated with (HS; 1-h HS plus 4-h
recovery) or without (non-HS) HS. The activation of indicated luciferase reporters was determined by dual-luciferase assays of the lysates. Data are
expressed as fold increase relative to untreated control RNAi cells and presented as mean ⫾ SD of triplicate samples. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
TLR4-triggered, Myd88-dependent and Toll/IL-1R domain-containing adapter inducing IFN␤ (TRIF)-dependent signaling pathways in tumor cells was attenuated by TLR4 silencing.
and B), suggesting that the released HSP70 from heat-stressed
tumor cells activates DC through TLR4.
Released HSP70 from heat-stressed tumor cells activates DC
through TLR4 in a paracrine manner
HSP70 release and TLR4 expression are both required for
HT-induced local and systemic activation of DC
As observed above, DC in the tumor tissue and spleen could be
activated by local HT (Figs. 1–3). Therefore, we examined the
in vitro effects of SN derived from HS-treated tumor cells on
DC. We found that the SN derived from HS-treated 3LL cells
could promote the phenotypic maturation and cytokine production of DC, whereas the supernatants from HSP70 (both HSC70
and HSP70i)-silenced, HS-treated 3LL cells showed the impaired capacity in promoting maturation and cytokine production of DC (Supplemental Fig. S7, A and B), suggesting that
HSP70 released from HS-treated tumor cells was essential for
the activation of DC via paracrine manner. To further elucidate
the roles of TLR4 in activation of the chemoattracted DC by
HS-treated tumor cells, we prepared DC from TLR4⫺/⫺ mice.
We found that the SN derived from HS-treated 3LL cells
showed decreased capacity in induction of maturation and cytokine production of TLR4⫺/⫺ DC (Supplemental Fig. S8, A
To confirm the roles of HSP70 release and TLR4 expression in the
HT-induced infiltration, activation of DC and induction of CD8⫹
CTL response, we first observed the tumor growth and DC infiltration in the mice bearing HSP70 (both HSC70 and HSP70i)- or
TLR4-silenced 3LL cells after HT treatments. The tumor growth
inhibition by HT was impaired in the mice-bearing HSP70-silenced or TLR4-silenced 3LL cells (data not shown). We found
that the percentage of DC within the TIMC was significantly reduced in HSP70-silenced and TLR4-silenced 3LL tumors than
those in corresponding scrambled control 3LL tumors (Fig. 7A),
suggesting that HSP70 release and TLR4 expression in tumor cells
were both required for the initial infiltration of DC into tumor
tissue after HT.
To elucidate the effects of released HSP70 in activation of DC,
we analyzed the phenotype and chemokine receptor expression of
DC enriched from HSP70-silenced 3LL tumor nodules. We found
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1457
To examine the effects of TLR4 expression in chemoattraction and activation of DC, we inoculated parental 3LL tumor
cells in TLR4⫺/⫺ knockout mice. We found that the tumor infiltration of DC in TLR4-deficient mice was slightly but not
significantly decreased before and after HT (data not shown).
However, these tumor-infiltrating DC in TLR4-deficient mice
were found to be impaired in their phenotypic maturation (Supplemental Fig. S9B) and chemokine receptor CCR7 and CXCR4
expression (Supplemental Fig. S10B) promoted by HT. Correspondingly, the DC in spleen and lymph nodes of TLR4⫺/⫺
mice bearing parental 3LL tumor after HT were significantly
decreased (Fig. 7C).
Finally, to show the importance of releasable HSP70 in the
induction of specific antitumor immunity, we analyzed the
CD8⫹ CTL induction in the mice-bearing HSP70 (both HSC70
and HSP70i)-silenced 3LL tumor. We found that the IFN-␥ production and cytotoxicity of CD8⫹ T cells were both decreased,
as compared with that in the mice-bearing scrambled control
3LL tumor (Fig. 7, D and E). These data further demonstrated
the important roles of releasable HSP70 in the induction of
specific CD8⫹ CTL.
Discussion
FIGURE 7. HSP70 release and TLR4 expression are both required for
HT-induced infiltration and activation of DC. A, FACS assays of DC infiltration. Mice bearing HSP70 (both HSC70 and HSP70i)-, TLR4-, or
corresponding scrambled control (Ctrl RNAi) vector-silenced 3LL tumors
were treated with (HT) or without (pre-HT) local HT for 1 h. Eight hours
later, the TIMC within tumor nodules were isolated. The infiltrated DC
were analyzed by FACS for CD11c⫹ cells. The results were expressed as
percentage of CD11c⫹ cells to that of TIMC, and the data were presented
as mean ⫾ SEM of five mice. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01. B and C, FACS
assays of DC within spleen (left panels) and lymph nodes (right panels).
Mice used in A were treated with (HT; 1-h HT plus 24-h recovery) or
without (pre-HT) HT. Then, DC within spleen and lymph nodes were examined by FACS for CD11c⫹ cells. Results were expressed as percentage
of CD11c⫹ cells to that of mononuclear cells, and the data were presented
as mean ⫾ SEM of five mice in each group. Œ, p ⬎ 0.05; ⴱ, p ⬍ 0.05; ⴱⴱ,
p ⬍ 0.01. D and E, In vitro induction of CTL cells. 3LL cells stably
silenced with scrambled control vector (Ctrl RNAi) or HSP70 (for both
HSC70 and HSP70i) silencing vector (HSP70 RNAi) were inoculated in
wild-type C57BL/6 mice. CD11c⫹ and CD8⫹ cells were isolated from
splenocytes derived from HT-treated mice (1-h HT plus 24-h recovery) by
magnetic beads and cocultured in vitro for 7 days. D, The cocultured CD8⫹
cells were stimulated with MUT1-pulsed matured BMDC and analyzed for
IFN-␥ release by ELISA. E, The cocultured cells were examined for cytotoxicity by using MUT1-pulsed (MUT1-DC) or OVA (257–264)-pulsed
(OVA-DC) mature BMDC as targets. Results were presented as mean ⫾
SD of triplicate samples. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
that HSP70 (both HSC70 and HSP70i) silence decreased the HTinduced phenotypic maturation (Supplemental Fig. S9A) and
CXCR4/CCR7 expression of the tumor-infiltrating DC (Supplemental Fig. S10A). And the DC percentages in the spleen and
lymph nodes of mice-bearing HSP70-silenced tumors were also
decreased (Fig. 7B).
Various HSP were released from tumor cells upon HS treatment
(3–5). However, we found that HSP70 (including HSP70i and
HSC70) may be the major HSP responsible for the autocrine induction of chemokines by tumor cells, as evidenced by our siRNA
experiments showing that HSP70 silence, but not HSP90, or gp96
or HSP60 silence, could significantly impair the induction of chemokines and the subsequent chemoattraction of DC by tumor cells.
Moreover, the HSP70 (both HSC70 and HSP70i)-silenced 3LL
tumor demonstrated less DC infiltration and impaired phenotypic/
functional maturation of the tumor-infiltrating DC, indicating that
HT-induced infiltration of DC and other immune cell populations
may be due to the tumor-released HSP70, and HSP70 released by
tumor cells after HT may be prominent in the activation of infiltrated DC. We also have provided evidence that DC infiltrated
within tumor after HT showed the elevated expression of CXCR4
and CCR7, indicating that HT may induce DC maturation and
promote the migration of DC from tumor tissues to secondary
lymphoid organs. Twenty-four hours after HT, DC in the spleen
and lymph nodes were increased and showed the improved capacity in the induction of tumor Ag-specific CTL, which was confirmed by our FACS assays of DC within spleen and lymph nodes
and by our in vitro CTL induction assays. The major involvement
of HSP70, but not HSP90 and HSP60, in HT-induced chemoattraction and activation of DC may be due to the differential levels
of these HSP released by tumor cells and the different efficiency of
these HSP in activating target cells (1, 2, 5, 12, 35). When HSC70
was silenced alone, the chemokine production by 3LL cells after
HS was much lower than that of HSP70i-silenced 3LL cells,
strongly suggesting that the levels of HSP released by tumor cells
may significantly affect the relative contribution of each HSP to
production of chemokines during HS (data not shown).
It has been reported that HSP90, HSP70, and gp96 derived from
tumor cells may contain tumor Ags and can present the associated
Ags to APC, finally resulting in Ag-specific CTL induction (6). In
our studies, we did not examine the HSP70-, HSP90- and gp96chaperoned Ags released from tumor cells after HT, which suggested that these HSP may be capable of presenting tumor Ags to
DC, given that releasable HSP70 can increase chemokine production by tumor cells and promote the infiltration, activation, and
migration of DC. Therefore, our studies have suggested a potential
mechanism for the initiation of antitumor immunity during the HT
1458
RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4
treatments. HT may induce the release HSP70 (including HSC70
and HSP70i) from tumor cells, which can activate tumor cells via
autocrine manner for chemokine production and simultaneously
activate the chemoattracted DC via paracrine manner, finally leading to the uptake of tumor Ags chaperoned by HSP and the subsequent presentation of tumor Ags to naive T cells in spleen and
lymph node once the DC mature and migrate to spleen and lymph
nodes. Considering that local HT treatments were administered
repeatedly, we suggest that the autocrine and paracrine actions of
releasable HSP70 may be positively augmented, leading to the
observed inhibition of tumor growth and induction of antitumor
immunity. Whether or not other HSP, such as HSP90, gp96, and
HSP60, are also involved in the induction of chemokines and activation of DC needs further investigation.
HSP can bind and activate target cells via HSP receptors. Many
molecules have been suggested as receptors for HSP, including
TLR2/4, CD91, CD40, CD14, and so on (25–34). In our study, we
have identified TLR4 as the major receptor for HSP70 in the induction of chemokines and activation of DC both in vitro and in
vivo. It has been suggested that HSP70-induced proinflammatory
cytokine production is mediated via the MyD88/IL-1R-associated
kinase/NF-␬B signal transduction pathway and that HSP70 uses
both TLR2 and TLR4 to transduce its proinflammatory signal in a
CD14-dependent fashion (25, 26, 29, 31). We found that silence of
CD91 and CD40 expression in 3LL cells didn’t affect the induction
of CXCL10 after HS treatments, whereas TLR4 and CD14 silence
could affect the chemokine induction. TLR4 silence almost eliminated HS-induced activation of NF-␬B and IRF3 signaling pathways, suggesting that TLR4 may play a major role in autocrine
activation of tumor cells by HSP70, and CD14 may help the TLR4
signal transduction in a similar manner to those reported for LPS
recognition by macrophages (25, 27). One inconsistency of our
findings is the exclusion of TLR2 in mediating the signaling by the
released HSP70. However, we have shown that 3LL cells are negative for TLR2 expression, and HS treatments can activate the
TRIF-dependent activation of IRF3. Previous studies have suggested that activation of TLR2 induces the expression of CXCL8,
CCL5, CCL3, and CCL4, whereas activation of TLR4 induces the
expression of CXCL10, CCL5, CCL3, and CCL4 (22, 36). Therefore, TLR4, but not TLR2, is responsible for the releasable HSP70induced tumor cell production of chemokines in autocrine manner
after HT treatment by activating both MyD88- and TRIF-dependent signaling pathways.
In sum, we have demonstrated the important roles of releasable
HSP from heat-stressed tumor cells in the initiation of antitumor
immunity. Our studies suggest that HSP70 (including HSC70 and
HSP70i) released from tumor cells after HT is the major factor
responsible for subsequent autocrine induction of chemokines and
chemoattraction of DC and for the paracrine activation of DC via
TLR4 signaling pathways. We have also provided evidence for the
importance of TLR4 in the tumor immunotherapy. Our data suggest that TLR4, under certain conditions (e.g., HT), can favorably
elicit antitumor immunity through facilitating chemokine production by tumor cells and through promoting Ag presentation by DC.
Thus, our study provides a potential explanation for the mechanisms involved in HT-induced antitumor immunity.
Acknowledgments
We appreciate Drs. Qiuyan Liu and Minghui Zhang for helpful discussion
and Yan Li, Ting Zhang, and Mei Jin for their excellent technical
assistance.
Disclosures
The authors have no financial conflict of interest.
References
1. Wallin, R. P., A. Lundqvist, S. H. More, A. von Bonin, R. Kiessling, and
H. G. Ljunggren. 2002. Heat shock proteins as activators of the innate immune
system. Trends Immunol. 23: 130 –135.
2. Tsan, M. F., and B. Gao. 2004. Cytokine function of heat shock proteins.
Am. J. Physiol. 286: C739 –C744.
3. Johnson, J. D., and M. Fleshner. 2006. Releasing signals, secretory pathways, and
immune function of endogenous extracellular heat shock protein 72. J. Leukocyte
Biol. 79: 425– 434.
4. Asea, A. 2006. Initiation of the immune response by extracellular Hsp72: chaperokine activity of Hsp72. Curr. Immunol. Rev. 2: 209 –215.
5. Pockley, A. G. 2003. Heat shock proteins as regulators of the immune response.
Lancet 362: 469 – 476.
6. Srivastava, P. 2002. Interaction of heat shock proteins with peptides and antigen
presenting cells: chaperoning of the innate and adaptive immune responses. Annu.
Rev. Immunol. 20: 395– 425.
7. Baronzio, G., A. Gramaglia, and G. Fiorentini. 2006. Hyperthermia and immunity: a brief overview. In Vivo 20: 689 – 695.
8. Milani, V., and E. Noessner. 2006. Effects of thermal stress on tumor antigenicity
and recognition by immune effector cells. Cancer Immunol. Immunother. 55:
312–319.
9. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda,
and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are
hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162: 3749 –3752.
10. Chen, T., J. Guo, M. Yang, M. Zhang, W. Chen, Q. Liu, J. Wang, and X. Cao.
2004. Cyclosporin A impairs dendritic cell migration by regulating chemokine
receptor expression and inhibiting cyclooxygenase-2 expression. Blood 103:
413– 421.
11. Gao, B., and M. F. Tsan. 2003. Endotoxin contamination in recombinant human
heat shock protein 70 (Hsp70) preparation is responsible for the induction of
tumor necrosis factor ␣ release by murine macrophages. J. Biol. Chem. 278:
174 –179.
12. Wan, T., X. Zhou, G. Chen, H. An, T. Chen, W. Zhang, S. Liu, Y. Jiang, F. Yang,
Y. Wu, and X. Cao. 2004. Novel heat shock protein Hsp70L1 activates dendritic
cells and acts as a Th1 polarizing adjuvant. Blood 103: 1747–1754.
13. Guo, J., J. Zhu, X. Sheng, X. Wang, L. Qu, Y. Han, Y. Liu, H. Zhang, L. Huo,
S. Zhang, et al. 2007. Intratumoral injection of dendritic cells in combination with
local hyperthermia induces systemic antitumor effect in patients with advanced
melanoma. Int. J. Cancer 120: 2418 –2425.
14. Guo, J., M. Zhang, B. Wang, Z. Yuan, Z. Guo, T. Chen, Y. Yu, Z. Qin, and
X. Cao. 2003. Fractalkine transgene induces T cell-dependent antitumor immunity through chemoattraction and activation of dendritic cells. Int. J. Cancer 103:
212–220.
15. Wang, Y., T. Chen, C. Han, D. He, H. Liu, H. An, Z. Cai, and X. Cao. 2007.
Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood 110:
962–971.
16. Bouchier-Hayes, L., H. Conroy, H. Egan, C. Adrain, E. M. Creagh,
M. MacFarlane, and S. J. Martin. 2001. CARDINAL, a novel caspase recruitment
domain protein, is an inhibitor of multiple NF-␬B activation pathways. J. Biol.
Chem. 276: 44069 – 44077.
17. Shinobu, N., T. Iwamura, M. Yoneyama, K. Yamaguchi, W. Suhara,
Y. Fukuhara, F. Amano, and T. Fujita. 2002. Involvement of TIRAP/MAL in
signaling for the activation of interferon regulatory factor 3 by lipopolysaccharide. FEBS Lett. 517: 251–256.
18. Dai, S., T. Wan, B. Wang, X. Zhou, F. Xiu, T. Chen, Y. Wu, and X. Cao. 2005.
More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared
from heat-stressed CEA-positive tumor cells. Clin. Cancer Res. 11: 7554 –7563.
19. Dai, S., X. Zhou, B. Wang, Q. Wang, Y. Fu, T. Chen, T. Wan, Y. Yu, and X. Cao.
2006. Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8⫹ CTL response by exosomes derived from IL-18
gene-modified CEA-positive tumor cells. J. Mol. Med. 84: 1067–1076.
20. Tirosh, B., K. el-Shami, N. Vaisman, L. Carmon, E. Bar-Haim, E. Vadai,
M. Feldman, M. Fridkin, and L. Eisenbach. 1999. Immunogenicity of H-2Kb-low
affinity, high affinity, and covalently-bound peptides in antitumor vaccination.
Immunol. Lett. 70: 21–28.
21. El-Shami, K. M., B. Tirosh, D. Popovic, L. Carmon, E. Tzehoval, E. Vadai,
M. Feldman, and L. Eisenbach. 2000. Induction of antitumor immunity by proteasome-inhibited syngeneic fibroblasts pulsed with a modified TAA peptide. Int.
J. Cancer. 85: 236 –242.
22. Luster, A. D. 2002. The role of chemokines in linking innate and adaptive immunity. Curr. Opin. Immunol. 14: 129 –135.
23. Balkwill, F. 2003. Chemokine biology in cancer. Semin. Immunol. 15: 49 –55.
24. Moser, B., M. Wolf, A. Walz, and P. Loetscher. 2004. Chemokines: multiple
levels of leukocyte migration control. Trends Immunol. 25: 75– 84.
25. Asea, A., S. K. Kraeft, E. A. Kurt-Jones, M. A. Stevenson, L. B. Chen,
R. W. Finberg, G. C. Koo, and S. K. Calderwood. 2000. Hsp70 stimulates cytokine production through a CD-14-dependent pathway, demonstrating its dual
role as a chaperone and cytokine. Nat. Med. 6: 435– 442.
26. Asea, A., M. Rehli, E. Kabingu, J. A. Boch, O. Bare, P. E. Auron,
M. A. Stevenson, and S. K. Calderwood. 2002. Novel signal transduction pathway utilized by extracellular HSP70: role of Toll-like receptor (TLR) 2 and
TLR4. J. Biol. Chem. 277: 15028 –15034.
The Journal of Immunology
27. Kol, A., A. H. Lichtman, R. W. Finberg, P. Libby, and E. A. Kurt-Jones. 2000.
Cutting edge: heat shock protein (HSP) 60 activates the innate immune response—CD14 is an essential receptor for HSP60 activation of mononuclear
cells. J. Immunol. 164: 13–17.
28. Vabulas, R. M., P. Ahmad-Nejad, C. da Costa, T. Miethke, C. J. Kirschning,
H. Hacker, and H. Wagner. 2001. Endocytosed HSP60s use Toll-like receptor 2
(TLR2) and TLR4 to activate the Toll/interleukin-1 receptor signaling pathway in
innate immune cells. J. Biol. Chem. 276: 31332–31339.
29. Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels, and
H. Wagner. 2002. Hsp70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277: 15107–15112.
30. Vabulas, R. M., S. Braedel, N. Hilf, H. Singh-Jasuja, S. Herter, P. Ahmad-Nejad,
C. J. Kirschning, C. Da Costa, H. G. Rammensee, H. Wagner, and H. Schild.
2002. The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J. Biol. Chem. 277:
20847–20853.
1459
31. Dybdahl, B., A. Wahba, E. Lien, T. H. Flo, A. Waage, N. Qureshi,
O. F. Sellevold, T. Espevik, and A. Sundan. 2002. Inflammatory response after
open heart surgery: release of heat shock protein 70 and signaling through Tolllike receptor-4. Circulation 105: 685– 690.
32. Becker, T., F. U. Hartl, and F. Wieland. 2002. CD40, an extracellular receptor for
binding and uptake of Hsp70-peptide complexes. J. Cell Biol. 158: 1277–1285.
33. Binder, R. J., D. K. Han, and P. K. Srivastava. 2000. CD91: a receptor for heat
shock protein gp96. Nat. Immunol. 1: 151–155.
34. Basu, S., R. J. Binder, T. Ramalingam, and P. K. Srivastava. 2001. CD91 is a
common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin.
Immunity 14: 303–313.
35. Bethke, K., F. Staib, M. Distler, U. Schmitt, H. Jonuleit, A. H. Enk, P. R. Galle,
and M. Heike. 2002. Different efficiency of heat shock proteins (HSP) to activate
human monocytes and dendritic cells: superiority of HSP60. J. Immunol. 169:
6141– 6148.
36. Re, F., and J. L. Strominger. 2001. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells. J. Biol. Chem. 276: 37692–37699.