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The Journal of Immunology

Heat Shock Protein 70, Released from Heat-Stressed TumorCells, Initiates Antitumor Immunity by Inducing Tumor CellChemokine Production and Activating Dendritic Cells viaTLR4 Pathway1

Taoyong Chen,2Jun Guo,2,3Chaofeng Han, Mingjin Yang, and Xuetao Cao4

Extracellular heat shock proteins (HSP) can activate dendritic cells (DC) and monocytes/macrophages, and HSP derived fromtumor 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 fullyelucidated. In this study, we report that heat stress can induce the release of various HSP from tumor cells, which, in turn, activatetumor cells to produce chemokines for chemoattraction of DC and T cells via TLR4 signaling pathway. In vivo, we find that theinfiltration and function of DC and T cells within tumor after local hyperthermia are increased significantly. We also provideevidence that HSP70 proteins released by tumor cells and TLR4 expressed by tumor cells/DC are essential for the chemoattractionof DC/T cells and for the subsequent induction of tumor-specific antitumor immunity. Therefore, our study suggests that heatstress-induced releasable HSP70 proteins from tumor cells play important roles in the initiation of antitumor immunity byinducing tumor cell production of chemokines and by activating the chemoattracted DC via TLR4 pathway. The Journal ofImmunology, 2009, 182: 1449 –1459.

Traditionally, heat shock proteins (HSP)5are regarded aschaperones assisting protein folding and translocation.However, HSP can also serve as cytokines that can stim-

released during cell injury conditions, such as surgery, excessiveexercise, and necrosis) and active (e.g., translocation of HSP toplasma membrane and subsequent secretion) pathways (3–5).

ulate dendritic cells (DC) and macrophages to produce proinflam-matory cytokines and chemokines (1–5). More importantly, HSPderived from tumor cells are capable of chaperoning tumor Ags toDC and then cross-presenting the Ags to T cells (6). HSP70 pro-teins, including the constitutively expressed cognate HSP70(HSC70 or HSP73), the stress-inducible HSP70 (HSP70i orHSP72), and the mitochondrial HSP70 (HSP75), constitute themost conserved class of all HSP. Previous reports have shown thatHSP can be released from various cells via passive (e.g., HSP

National Key Laboratory of Medical Immunology and Institute of Immunology, Sec-ond 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 accordancewith 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 Foun-dation of China (30572122, 30771118, and 30721091), National Key Basic ResearchProgram of China (2007CB512403), and Shanghai Committee of Science and Tech-nology (07QA14067).

2 T.C. and J.G. contributed equally to this work.

3 Current address: Department of Renal Cancer and Melanoma, Beijing Cancer Hos-

pital and Institute, Beijing, People’s Republic of China.

4 Address correspondence and reprint requests to Dr. Xuetao Cao, National Key Lab-

oratory of Medical Immunology and Institute of Immunology, Second Military Med-ical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China.E-mail address: [email protected]

5 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-celllysate; TRIF, Toll/IL-1R domain-containing adapter inducing IFN .

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org

However, the roles of HSP70 proteins released from tumor cells inthe induction of antitumor immunity and the underlying mecha-nisms have not been fully elucidated.

Hyperthermia (HT) has been reported to enhance the immu-nogenecity of cancer cells concomitantly with expression ofHSP (7, 8). Recent reports demonstrate that heat stress (HS) caninduce 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 initiateantitumor immunity via released HSP and via subsequent acti-vation of DC still lack direct evidence and thus need to befurther investigated.

During the investigations of local HT (42– 43°C)-elicited an-titumor immunity, we find that infiltration of DC and T cellswithin heat-stressed tumor is significantly increased. We thushypothesize that chemokines, induced by HS, may be involvedin the initiation of HT-elicited antitumor immunity by chemoat-traction and activation of DC. Our studies show that HSP70proteins simultaneously released by tumor cells can serve asautocrine and paracrine cytokines inducing the production ofvarious chemokines by tumor cells and the activation of DC viaTLR4 signaling pathway. Our data provide direct evidence forthe important roles of releasable HSP in the initiation of anti-tumor immunity during local HT.

Materials and MethodsMice, cells, Abs, and reagentsMale wild-type C57BL/6 (H-2Kb) mice, 6 – 8 wk of age, were obtainedfrom SIPPR-BK Experimental Animal Company. TLR4 knockout mice(TLR4/,C57BL/6 strain) were provided by Dr. S. Akira (Research In-stitute for Microbial Diseases, Osaka, Japan) (9). All mice were housed ina specific pathogen-free facility for all experiments. Murine Lewis lungcarcinoma 3LL and murine malignant melanoma B16 cells were obtained

1450 RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4

from American Type Culture Collection. These cell lines were maintainedin appropriate medium as recommended. Mouse bone-marrow derived DC(BMDC) were prepared by culturing with 20 ng/ml rGM-CSF and 10ng/ml IL-4 (Genzyme) as described previously (10). The mAbs againstHSP70i, 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 fortotal and phosphorylated forms of IFN regulatory factor 3 (IRF3; Ser 396)and I B (Ser32/36) were obtained from Cell Signaling Technology. Flu-orescent Abs against CD80, CD86, Iab, CD40, CD11c, CD3, CD4, CD8,CD25, DX5, Gr1, FoxP-3, and isotype control Abs were obtained from BDPharmingen. The rHSP proteins were further deprived of LPS contamina-tion as described by using polymycin B agarose (Sigma-Aldrich) (11). Thepurity ( 92%) and LPS contamination ( 5 pg/ g protein) of rHSP weredetermined as described previously (12). ELISA kits for measuring che-mokines and cytokines were purchased from R&D Systems. Other non-specified reagents were purchased from Sigma-Aldrich.

Establishment and monitor of tumor model, local HT treatments,and immunological assessment of systemic antitumor immunity3LL cells (5 105) in 100 l of PBS were s.c. injected into the shavedright flanks of the C57BL/6 mice (H-2Kb). Seven days after the inoc-ulation, the tumors (100 –150 mm3) were treated by using a 915-MHzmicrowave machine for superficial tumor at 42– 43°C for 1 h at aninterval of 1 wk for a total of three times. Body surface temperature wasmaintained at 42– 43°C, which was confirmed by a thermosensor duringHT for superficial tumors as previously described (13), and could in-duce significant increase of HSP expression as determined by Westernblot assays. Tumor sizes were measured every 2–3 days, and the tumorvolumes were determined by measuring of the maximal (a) and minimal(b) diameters using a caliber and calculated by using the formula ab2. The survival of the tumor-bearing mice was observed daily as de-scribed previously (14). Mice were sacrificed when the tumors reached2 cm in diameter or appeared moribund, and this was recorded as thedate of death for survival studies.

For assessment of systemic CTL induction, 12 days after the lasttreatment of tumor with HT, CTL activity was measured by using astandard 4-h51Cr release assay as described previously (14). 3LL cellswere used as targets and syngeneic lymphoma EL-4 cells were used ascontrol targets.

Immunohistochemistry

Different hours after the first treatment of HT, tumors were isolated, frozensectioned, and examined for the infiltrations of DC and T cells as describedpreviously (14). The number of immunostained cells was examined bylight microscopy.

Isolation of tumor-infiltrating mononuclear cells, DC, andT cellsFor the isolation of tumor-infiltrating mononuclear cells (TIMC), tumortissues were cut into small fragments and incubated in RPMI 1640 mediumcontaining 1 mg/ml collagenase (4 ml/g tissue) and 0.25% DNase I at 37°Cfor 45 min as described previously (10). Then, single-cell suspensions ofTIMC, lymph node mononuclear cells, and splenocytes were prepared andcollected by centrifugation on a Ficoll gradient. For the isolation of DC andT cell populations, the isolated immune cell suspensions were incubatedwith magnetic beads specifically for CD11c , CD3 , CD4 , or CD8markers and then isolated by immunomagnetic separation (MACS; Milte-nyi 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 106cellsin 5 ml medium) were heat treated at 42°C in an air incubator contain-ing 5% CO2.

In vitro chemoattraction assay

To evaluate the chemotactic activity of culture supernatants (SN) fromtumor cells, chemotaxis of mouse DC and splenic T cells was assayed asdescribed previously by us (10).

RT-PCR and quantitative PCR

Total cellular RNA was extracted using TRIzol reagent (Invitrogen). RT-PCR was performed as described previously (10). Specific primers used forRT-PCR assays of chemokines, chemokine receptors (CXCR4 and CCR7),and HSP receptors (TLR4, TLR2, CD40, CD14, and CD91) were available

upon request. Quantitative PCR was performed on a MJR Chromo4 Con-tinuous Fluorescence detector (Bio-Rad) according to the manufacturer’sprotocol and as described previously (15).

Western blotting

Total cell lysates were prepared as described previously, and protein con-centration was determined by the bicinchoninic acid protein assay (Pierce).Cell extracts were subjected to SDS-PAGE, transferred onto nitrocellulosemembrane, 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 CAUCGC CAA CUU-3 (sense) and 5 -GUU GGC GAU GAU CUC CACCUU-3 (antisense) for HSP70i; 5 -UGA ACC CCA CCA ACA CAGUUU-3 (sense) and 5 -ACU GUG UUG GUG GGG UUC AUU-3 (an-tisense) for HSC70; 5 -ACC CAG ACC CAA GAC CAA CUU-3 (sense)and 5 -GUU GGU CUU GGG UCU GGG UUU-3 (antisense) forHSP90 ; 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 CCAACU GAA UAG CUU CUU-3 (antisense) for gp96; and 5 -UGC UUCAAG GUG UAG ACC UUU-3 (sense) and 5 -AGG UCU ACA CCUUGA AGC AUU-3 (antisense) for HSP60. The corresponding sequencescontaining two nucleotide mutations were used as a scrambled control(Ctrl). All the used sequences were blasted against the National Center forBiotechnology Information nucleotide database to exclude nonspecificinterference. siRNA duplexes were transfected into tumor cells usingINTERFERin reagent (Polyplus-transfection) according to the standardprotocol and as described previously (15). For exclusion of the possibleoff-targets effects of HSP70i siRNAs, HSP70i sequence containing twosynonymous mutations (G84 to C and C96 to G, -HSP70i) in pcDNA3.1vector (Invitrogen) was transfected into 3LL cells.

For stable knockdown of TLR4, CD91, CD40, and CD14 expression in3LL tumor cells, an expression vector (psilencer-U6 neo; Ambion) withinsertion of specific siRNA duplexes of the indicated receptors or the cor-responding scrambled siRNA duplexes were transfected into 3LL cellsusing Jetpei (Polyplus-transfection) according to the standard protocol andas described previously (15). The targeting 19-nt sequences used were asfollows: 5 -GCT TGA ATC CCT GCA TAG A-3 for TLR4, 5 -CTT CTCAGA TCC GAA GCC A-3 for CD14, 5 -AAC TTG CAG CCC TAAGCA G-3 for CD91, and 5 -GCC GAC TGA CAA GCC ACT G-3 forCD40. For stable silence of both HSP70i and HSC70, we used the se-quences targeting 5 -TCC TAT GCC TTC AAC ATG A-3 for construc-tion of RNAi vectors, which was less efficient than that used for transientsilence and could lead to 70% down-regulation of both HSP70i andHSC70 proteins. After transfection of the RNAi vectors, cells were se-lected 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 againstI B and IRF3 were used to detect the expression levels of these mole-cules in whole-cell lysates (WCL) by Western blot. To examine the nucleartranslocation of NF- B and IRF3, the extracted nuclear proteins, usingNE-PER nuclear reagents (Pierce), were Western blotted to detect the pres-ence of NF- B p65 subunit and IRF3 in the nucleus as described previ-ously (10, 15).

Luciferase reporter assay

The determination of NF- B and IRF3 transactivation was performed asdescribed previously (15). The pGL3.5X B-luciferase reporter plasmidwas provided by S. J. Martin (Trinity College, Dublin, Ireland) (16). TheIRF3 reporter plasmids were gifts from Dr. T. Fujita (Tokyo MetropolitanInstitute of Medical Science, Tokyo, Japan) (17). The pRL-TK-Renilla-luciferase plasmid was obtained from Promega.

Functional assessment of DC

To evaluate the functional status of DC isolated from tumor tissues (en-riched from TIMC with CD11c-specific magnetic beads), DC were sub-jected to the assays of phenotype, cytokine secretion assay, and allogeneicstimulatory capacity assay as described previously (10).

Functional assessment of tumor-infiltrating T cells

To evaluate the functional status of tumor-infiltrating T cells, CD3 cellsisolated from TIMC using immunomagnetic beads were cultured in vitro


FIGURE 1. Local hyperthermiapromotes the infiltration of DC and Tcells in vivo. A, FACS assays of in-filtrated DC (CD11c ), T cells(CD3 CD4 or CD3 CD8 ) andNK cells (DX5 ) within the TIMCpopulations of 3LL tumors treatedwith (3LL HT) or without (3LL) HTfor 1 h and recovered for 8 h. Theresults were representative imagesobtained from five mice, and thenumbers indicated for percentages ofcells to all the TIMC populations. B,FACS analysis of Treg and MDSCpopulations. The isolated TIMC cellswere purified for CD4 and CD11bpopulations and then stained forFoxP-3/CD25 and Gr1 as indicated.Numbers indicated for percentagesCD25 FoxP-3 (upper panel) andGr1 (lower panel) cells to all theCD4 and CD11b cells respec-tively. 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 tu-mors treated with (1-h HT plus 4-, 8-,12-, and 24-h recovery, respectively)or without (0 h) HT. Results werepresented as percentages of indicatedcells to total TIMC and expressed asmean SEM of five mice. , p0.05; , p 0.01; , p 0.001.

and stimulated with 2 g/ml anti-CD3 and anti-CD28 mAb (BD Pharm-ingen) and then examined for the proliferation and cytokine productioncapacity as described previously (18, 19).

ResultsLocal HT increases the infiltration of DC and T cells but

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In vitro CTL induction and cytotoxicity assays

To evaluate the DC-T cell interaction within spleen after HT treatments invitro, enriched CD11c (2 105DC/well) and CD8 cells derived fromspleen of HT-treated mice were cocultured in vitro in medium containing30 U/ml IL-2 (PeproTech) for 7 days in 24-well plates in a final volume of2 ml/well (1 DC per 10 CD8 T cells). Every 2 days, 0.5 ml of supernatantwas replaced with fresh medium. Seven days later, functions of stimulatedCD8 T cells were evaluated by IFN- release assay and cytotoxicity assayas described previously (18, 19). To examine 3LL-specific CTL induction,we prepared BMDC from normal wild-type mice after in vitro cultured for5 days, matured them with 100 ng/ml LPS stimulation for 48 h, pulsed themwith 50 g/ml synthetic MHC-I-restricted peptides MUT1 (FEYNYAQL,high affinity for H-2Kb) and OVA (257–264, SIINFEKL, H-2Kb) at thefinal 1 h (20, 21), and irradiated (5000 rad) them. For IFN- release assay,stimulator cells (DC pulsed with MUT1) were cocultured with the stimu-lated CD8 T cells at different ratios. Forty-eight hours later, the SN weretested for the level of IFN- by ELISA. Cytotoxicity assays were per-formed using standard 4-h51Cr release assay against labeled target cells asdescribed (14, 19). Targets for CTL activity were mature DC pulsed withMUT1 or OVA peptides for 1 h.

Statistical analysis

All experiments were independently performed three times. Results aregiven as means SE or means SD. Comparisons between two groupswere done using Student’s t test, while comparisons between multiplegroups were done using Kruskal-Wallis tests. Survival estimates and me-dian survivals were determined using the method of Kaplan and Meier.Statistical significance was determined as values of p 0.05.

relatively decreases the infiltration of regulatory T cells (Treg)and myeloid-derived suppressor cells (MDSC) in the tumortissueAfter treatments of pre-established 3LL tumor with HT, we foundthat HT could inhibit the in vivo growth of 3LL Lewis lung car-cinoma (Supplemental Fig. S1A)6and prolong the survival of thetumor-bearing mice (Supplemental Fig. S1B). CTL assays indi-cated that HT could elicit potent 3LL tumor-specific antitumorimmunity (Supplemental Fig. S1C).

We then investigated the infiltration of immune cell populationswithin the tumor tissue by immunohistochemistry. We found thatthe tumor infiltrations of DC (CD11c ) and T cells (CD4 orCD8 ) at different time points after the first treatment of HT wereincreased significantly (Supplemental Fig. S2). To more accuratelyassess the infiltration of DC and T cells within tumor tissue afterHT, we isolated TIMC 8 h after the first HT treatment and ana-lyzed the cell populations within tumor by FACS. We found thatthe numbers of DC (CD11c ), T cells (CD3 CD4 and CD3CD8 ), and NK cells (NK, DX5 ) were significantly increasedafter HT (Fig. 1A). We further examined Treg (CD4 CD25FoxP3 ) and MDSC (Gr1 CD11b ) within tumor after HT. Wefound that the percentages of Treg and MDSC cells in TIMC were

6 The online version of this article contains supplemental material.


FIGURE 2. Local HT improves the functional status of infiltrated DC and T cells within tumor. TIMC populations were isolated from tumor tissue beforeHT (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 (1105/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 LPSor 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 (0h) 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 oftriplicate 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-CD3and anti-CD28 (anti-CD3/CD28) Ab as indicated. Then, proliferation was examined by [3H]thymidine incorporation assays (E), and cytokines in thesupernatants were examined by ELISA (F). Results were presented as mean SD of triplicate samples. , p 0.05; , p 0.01; , 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 firstHT, and we found that the infiltration of these populations of cellswas increased in total numbers after HT (data not shown), and thepercentages of immunosuppressive cell populations (MDSC andTreg) of TIMC were relatively decreased due to the increase ofinfiltrated cells (Fig. 1C), which may finally favor the observedinhibition of tumor growth and induction of tumor-specific CTL invivo (Supplemental Fig. S1). As further evidence, we treated B16

melanoma-bearing tumor models with local HT and found that HTcould inhibit tumor growth, improve survival and promote the in-filtration of DC and T cells at a similar extent to that observed in3LL tumor models (data not shown).

Local HT improves local and systemic activation of DC andT cellsWe then isolated the tumor-infiltrating DC and T cells from TIMCby magnetic beads specific for CD11c and CD3 cells, respec-tively, with purity 90%. We found that DC-derived from the


HT-treated tumor (8 h after the first HT treatment) showed ele-vated expression levels of surface Iab, CD80, CD86, and CD40(Fig. 2A). Moreover, such DC showed more potent capacity inproducing IL-12p70 and TNF- (Fig. 2B) and in stimulating theproliferation of allogeneic CD3 T cells (Fig. 2C). Also, theexpression of CCR7 and CXCR4 were significantly increased insuch DC (Fig. 2D). These data suggested that HT treatment notonly increased the numbers of infiltrated DC (Fig. 1) but alsoimproved the maturation and Ag presentation capacity of thesetumor-infiltrating DC.

To examine the functional status of tumor-infiltrating T cells, weincubated the enriched CD3 T cells with anti-CD3/CD28 Ab for48 h and then found that T cells derived from HT-treated tumorsshowed elevated capacity in proliferation (Fig. 2E) and increasedproduction of IL-2 and IFN- while decreased production ofTGF- and IL-10 (Fig. 2F). These data suggested that the func-tional status of T cells within tumors after HT was also improved.

Then, we examined the migration of DC and T cells in thespleen and draining lymph nodes of tumor-bearing mice after localHT. We found that local HT could significantly increase the per-centage of DC within these secondary lymphoid organs (Fig. 3A),suggesting that HT might promote DC to migrate to spleen and LNfor tumor Ag presentation. To test this possibility, we isolatedsplenic CD11c DC from 3LL tumor-bearing mice with or with-out HT treatments, cocultured them with splenic CD8 T cellsderived from 3LL tumor-bearing mice, and examined the CTLinduction. We found that splenic DC derived from HT-treated 3LLtumor-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 ofDC and CD8 T cells in the secondary lymphoid organs.

Heat-stressed tumor cells release chemokines and subsequentlychemoattract DC and T cells in vitroChemokines play essential roles in the regulation of DC residencyin peripheral tissues and DC migration to secondary lymphoid or-gans (22–24). Therefore, we hypothesized that local HT could pro-mote the production of chemokines by tumor cells. We found thatthe SN derived from HS-treated 3LL tumor cells could chemoat-tract more CD11c DC and CD3 T cells than that from untreated3LL cells (Fig. 4A). Similar results were observed when B16 mel-anoma and other tumor cell lines were used as alternative models(data not shown).

To examine whether HS could induce chemokine production in3LL cells, we performed RT-PCR assays of chemokines in 3LLtumor cells before and after HS treatments and found that HStreatments could significantly increase the expression of chemo-kines, including CC chemokines (CCL2/MCP-1, CCL3/MIP-1 ,CCL4/MIP-1 , CCL5/RANTES, CCL19/MIP-3 , CCL20/MIP-3 , and CCL25/thymus-expressed chemokine), CXC chemokine(IP-10), and CX3C chemokine (CX3CL1/fracktalkine) (Supple-mental Fig. S3). We examined the tumor cell production of CCL2,CCL5, and CXCL10 by both quantitative RT-PCR and ELISA andconfirmed that these chemokines were all significantly increasedby HS treatments (Fig. 4, B and C).

Releasable HSP70 derived from tumor cells after HS arerequired for the chemoattraction of DCUp-regulation of HSP is a typical effect of HS on cells, andreleasable HSPs have been shown to function as cytokines toactivate DC and macrophages (3, 4). We then hypothesized thatHS may induce chemokine expression of tumor cells by thereleased HSP.

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FIGURE 3. Local hyperthermia promotes the presence of DC withinspleen and lymph nodes and the induction of CTL. A, Percentages ofCD11c cells within spleen and lymph node. The 3LL tumor-bearing micewere treated for 1 h by local HT (3LL HT), and the spleen and lymph nodesof the mice were isolated for mononuclear cells 24 h after HT. Then, DC(CD11c ) in the mononuclear cells were analyzed by FACS assays, andthe results were representative data derived from five mice and presentedas 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. Thesplenic CD11c cells derived from HT-treated mice (3LL HT) or non-treated mice (3LL) were cocultured with splenic CD8 T cells derivedfrom spleens of nontreated 3LL tumor-bearing mice for 7 days at a ratio of1:10. 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/mlhigh-affinity MUT1 peptides at the last 1 h as stimulators. C, The maturedBMDC (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 atindicated ratios. The MUT1-specific cytotoxicity was examined by51Crrelease assay. Results were presented as mean SD of triplicate samples., p 0.05; , p 0.01.

To test this possibility, we first examined the release of HSPby 3LL cells after HS treatments. We found that HSP90, gp96,HSC70, HSP70i, and HSP60 were all rapidly released into ex-tracellular medium after HS treatments within 10 min (Fig. 5A).During the 1 h of HS treatment, HSP90, gp96, HSP70i, andHSP60 in the cell lysates were not significantly elevated at pro-tein level (data not shown), indicating that HS induced the ac-tive release of HSP. We then blocked the released HSP by using

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FIGURE 4. SN from heat-stressedtumor cells contain chemokines andcan chemoattract DC and T cells invitro. A, In vitro chemoattraction as-says of the SN derived from non-treated (3LL SN) or heat-treated (3LLHT SN) cells toward DC (left panel)and T cells (right panel). DC were invitro-cultured immature BMDC (day5), and T cells were splenocytes de-rived from wild-type C57BL/6 mice.Medium was used as negative con-trol. Results were presented asmean SD of triplicate samples. ,p 0.01. B and C, HS induces che-mokine production of 3LL cells. 3LLcells (0 h) were subjected to HS treat-ments at 42°C for 1 h and then recov-ered at 37°C for 4 and 8 h as indi-cated. Then, 3LL cells were used forquantitative RT-PCR assays of che-mokine production (B), and the SNwere measured for chemokine pro-duction by ELISA (C). Results werepresented as mean SD of triplicatesamples. , p 0.05; , p 0.01;

, p 0.001.

RELEASABLE HSP70 INITIATES ANTITUMOR IMMUNITY VIA TLR4

neutralizing Abs and performed the in vitro chemoattractionassays. We found that blocking Abs specific for HSC70 andHSP70i could significantly inhibit the capacity of HS-treated3LL SN to chemoattract DC (Supplemental Fig. S4A). And to alesser extent, blocking Abs for HSP90 and gp96, but notHSP60, could also inhibit the chemotactic activity of the HS-treated 3LL SN (Supplemental Fig. S4A). Similarly, supplementof rHSP into the SN of 3LL without HS treatments could elicitsimilar 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 respon-sible for the observed chemotactic activity of HS-treated 3LLSN toward DC.

To investigate which kind of HSP plays prominent roles in elic-iting chemotactic activity via inducing chemokine production, wesilenced the expression of HSP60, HSP70s, HSP90s, and gp96 viatransient transfection of siRNA (Supplemental Fig. S5). We foundthat the chemotactic activity of SN derived from HSP70 (bothHSC70 and HSP70i)-silenced cells after HS treatment toward DCdecreased significantly (Fig. 5B). Moreover, similar to those ob-served in Ab blocking experiments (Supplemental Fig. S4A),HSP90 and gp96 silence could also reduce the HS-treated 3LLSN-mediated chemoattraction of DC (to a much lesser extent),whereas HSP60 silence has no significant effects on the chemo-tactic activity toward DC (Fig. 5B).

Releasable HSP70 derived from tumor cells after HSup-regulates the expression of chemokines in tumor cells viaTLR4-mediated signaling pathwayWe next examined the roles of HSP70 in the induction of chemo-kine production by tumor cells during HS treatments. We found

that the HS-induced up-regulation of the chemokines’ mRNAwas significantly reduced in HSP70 (both HSC70 and HSP70i)-silenced 3LL cells, as compared with that in scrambled controlsiRNA-silenced 3LL cells (Ctrl RNAi; Fig. 5, C and D). Toexclude the possibility that the above decrease of chemotacticactivity by HSP70 silence was due to the roles of intracellularHSP70 during HS treatments, we supplemented rHSP70(HSC70 HSP70i) in the SN of HSP70-silenced 3LL cellsduring HS treatments. We found that the supplement of rHSP70proteins could rescue the observed reduction of DC chemoat-traction (Fig. 5B) and chemokine production by HSP70 silence(Fig. 5, C and D). For exclusion of possible off-targets ofHSP70 siRNA, we transiently transfected the HSP70-silenced3LL cells with -HSP70i vectors (resistant to HSP70 siRNA;Fig. 5E). We found that the overexpression of -HSP70i couldrescue the inhibitory effects of HSP70 siRNA on HS-inducedchemoattraction of DC and CXCL10 production (Fig. 5, F andG). These results suggested that the released HSP70 (includingHSP70i and HSC70) in the SN of HS-treated 3LL cells playedessential roles in the chemoattraction of DC and T cells byinducing tumor cell production of chemokines via an autocrinemanner.

Releasable HSP can activate target cells via receptor bindingand signaling (25–34). We thus examined the expression of TLR2,TLR4, CD40, CD91, and CD14 in 3LL tumor cells. We found thatTLR4, CD40, CD91, and CD14, but not TLR2, were all expressedby 3LL cells to a different extent, and HS treatments could elevatethe expression of these receptors (Supplemental Fig. S6A). Then,we wondered which receptor(s) was responsible for the inductionof chemokines by HSP70 during HS treatments. We thus stablysilenced the expression of CD91, CD14, CD40, and TLR4 in 3LLcells (Supplemental Fig. S6B) and then examined the induction of


FIGURE 5. HSP70 silence inhibits tumorcell chemokine production and subsequent che-moattraction of DC induced by HS. A, HS rap-idly induces the release of HSP into medium.3LL tumor cells were subjected to HS treat-ments (42°C) as indicated. Then, SN were col-lected by centrifugation at 13,000 g for 10min. HSP contained in the SN were examined byWestern blot as indicated. BSA supplemented inthe SN were probed as quantitative control. B,HSP silence affects the chemoattraction of SNtoward BMDC. 3LL cells silenced with siRNAtargeting HSP90, gp96, HSP70 (both HSC70and HSP70i), and HSP60 for 48 h were HStreated for 1 h and recovered for 8 h. Then, SNwere collected and subjected to in vitro che-moattraction assays. Results were expressed asnumber of CD11c cells that have migrated tolower chamber as counted by FACS and pre-sented as mean SD of triplicate samples. Œ,p 0.05; , p 0.001. C and D, HSP70silence inhibits HS-induced chemokine produc-tion. 3LL cells silenced with scrambled siRNAduplexes (ctrl siRNA) or HSC70/HSP70isiRNA (HSP70 siRNA) for 48 h were treatedwith HS for 1 h plus 4-h recovery (for quan-titative RT-PCR) or 8-h recovery (for ELISA)or treated without HS (non-HS). Then, cellsand SN were analyzed for mRNA (C) and pro-tein (D) expression of CXCL10, respectively.Results were presented as mean SD of trip-licate samples. Œ, p 0.05; , p 0.001.E, -HSP70i overexpression in HSP70-si-lenced cells. 3LL cells silenced with scram-bled siRNA duplexes (ctrl siRNA) or HSC70/HSP70i siRNA (HSP70 siRNA) weretransiently transfected with mock vector or

-HSP70i vector containing synonymous mu-tations resistant to HSP70 siRNAs and encod-ing HSP70i. WCL and SN treated with HS for1 h were examined by Western blot. F and G,

-HSP70i overexpression attenuates HSP70silence-induced inhibition of DC chemoattrac-tion (F) and chemokine production (G) afterHS (1-h HS plus 8-h recovery). Œ, p 0.05;

, p 0.001.

chemokines in these silenced 3LL cell by HS treatments. We foundthat TLR4-silenced 3LL cells showed significantly decreasedproduction of CXCL10, whereas the silence of CD14 also could re-duce the production of CXCL10 to some extent (Fig. 6, A andB). However, the silence of CD91 and CD40 did not affect theproduction of these chemokines (data not shown). Moreover,we found that TLR4 silence didn’t affect the HS-induced releaseof HSP70 and HSC70 by tumor cells (data not shown). How-ever, the SN derived from TLR4-silenced, HS-treated 3LL cellsshowed significantly decreased chemotactic activity toward DC(Fig. 6C).

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As further evidence, we examined the TLR4 signaling in TLR4-silenced 3LL cells after HS treatment. We found that the phos-phorylation of I B and the nuclear translocation of NF- B p65subunit were significantly reduced in TLR4-silenced 3LL cells ascompared with that in scrambled control siRNA-silenced 3LL cellsafter HS treatment (Fig. 6D). Moreover, the phosphorylation andnuclear translocation of IRF3 were also significantly reduced inTLR4-silenced 3LL cells (Fig. 6D). In addition, we found that bothNF- B and IRF3 gene reporter activation decreased significantlyby TLR4 silence during HS treatments of tumor cells (Fig. 6E).These data suggested that HS-induced activation of both


FIGURE 6. TLR4 silence inhibits tumor cell chemokine production and subsequent chemoattraction of DC induced by HS. A and B, Chemokineproduction 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) or8-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. , p 0.05; , p 0.001. C, In vitrochemoattraction 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 collectedfor SN. The chemoattraction of DC was expressed as number of CD11c cells that have migrated to lower chamber as counted by FACS andpresented as mean SD of triplicate samples. , p 0.001. D, Western blot assays of NF- B and IRF3 activation. The indicated cells were treatedwith 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 cellsstably silenced with TLR4 silencing vector (TLR4 RNAi) or scrambled control vector (Ctrl RNAi) were transiently transfected with luciferasereporter 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-hrecovery) or without (non-HS) HS. The activation of indicated luciferase reporters was determined by dual-luciferase assays of the lysates. Data areexpressed as fold increase relative to untreated control RNAi cells and presented as mean SD of triplicate samples. , p 0.05; , p 0.01.

TLR4-triggered, Myd88-dependent and Toll/IL-1R domain-con-taining adapter inducing IFN (TRIF)-dependent signaling path-ways in tumor cells was attenuated by TLR4 silencing.

Released HSP70 from heat-stressed tumor cells activates DCthrough TLR4 in a paracrine mannerAs observed above, DC in the tumor tissue and spleen could beactivated by local HT (Figs. 1–3). Therefore, we examined thein vitro effects of SN derived from HS-treated tumor cells onDC. We found that the SN derived from HS-treated 3LL cellscould promote the phenotypic maturation and cytokine produc-tion of DC, whereas the supernatants from HSP70 (both HSC70and HSP70i)-silenced, HS-treated 3LL cells showed the im-paired capacity in promoting maturation and cytokine produc-tion of DC (Supplemental Fig. S7, A and B), suggesting thatHSP70 released from HS-treated tumor cells was essential forthe activation of DC via paracrine manner. To further elucidatethe roles of TLR4 in activation of the chemoattracted DC byHS-treated tumor cells, we prepared DC from TLR4/mice.We found that the SN derived from HS-treated 3LL cellsshowed decreased capacity in induction of maturation and cy-tokine production of TLR4/DC (Supplemental Fig. S8, A

and B), suggesting that the released HSP70 from heat-stressedtumor cells activates DC through TLR4.

HSP70 release and TLR4 expression are both required forHT-induced local and systemic activation of DCTo confirm the roles of HSP70 release and TLR4 expression in theHT-induced infiltration, activation of DC and induction of CD8CTL response, we first observed the tumor growth and DC infil-tration in the mice bearing HSP70 (both HSC70 and HSP70i)- orTLR4-silenced 3LL cells after HT treatments. The tumor growthinhibition by HT was impaired in the mice-bearing HSP70-si-lenced or TLR4-silenced 3LL cells (data not shown). We foundthat the percentage of DC within the TIMC was significantly re-duced in HSP70-silenced and TLR4-silenced 3LL tumors thanthose in corresponding scrambled control 3LL tumors (Fig. 7A),suggesting that HSP70 release and TLR4 expression in tumor cellswere both required for the initial infiltration of DC into tumortissue after HT.

To elucidate the effects of released HSP70 in activation of DC,we analyzed the phenotype and chemokine receptor expression ofDC enriched from HSP70-silenced 3LL tumor nodules. We found


FIGURE 7. HSP70 release and TLR4 expression are both required forHT-induced infiltration and activation of DC. A, FACS assays of DC in-filtration. Mice bearing HSP70 (both HSC70 and HSP70i)-, TLR4-, orcorresponding scrambled control (Ctrl RNAi) vector-silenced 3LL tumorswere treated with (HT) or without (pre-HT) local HT for 1 h. Eight hourslater, the TIMC within tumor nodules were isolated. The infiltrated DCwere analyzed by FACS for CD11c cells. The results were expressed aspercentage of CD11c cells to that of TIMC, and the data were presentedas mean SEM of five mice. , p 0.05; , p 0.01. B and C, FACSassays 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) orwithout (pre-HT) HT. Then, DC within spleen and lymph nodes were ex-amined by FACS for CD11c cells. Results were expressed as percentageof CD11c cells to that of mononuclear cells, and the data were presentedas mean SEM of five mice in each group. Œ, p 0.05; , p 0.05; ,p 0.01. D and E, In vitro induction of CTL cells. 3LL cells stablysilenced with scrambled control vector (Ctrl RNAi) or HSP70 (for bothHSC70 and HSP70i) silencing vector (HSP70 RNAi) were inoculated inwild-type C57BL/6 mice. CD11c and CD8 cells were isolated fromsplenocytes derived from HT-treated mice (1-h HT plus 24-h recovery) bymagnetic beads and cocultured in vitro for 7 days. D, The cocultured CD8cells were stimulated with MUT1-pulsed matured BMDC and analyzed forIFN- release by ELISA. E, The cocultured cells were examined for cy-totoxicity by using MUT1-pulsed (MUT1-DC) or OVA (257–264)-pulsed(OVA-DC) mature BMDC as targets. Results were presented as meanSD of triplicate samples. , p 0.05; , p 0.01.

that HSP70 (both HSC70 and HSP70i) silence decreased the HT-induced phenotypic maturation (Supplemental Fig. S9A) andCXCR4/CCR7 expression of the tumor-infiltrating DC (Supple-mental Fig. S10A). And the DC percentages in the spleen andlymph nodes of mice-bearing HSP70-silenced tumors were alsodecreased (Fig. 7B).

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To examine the effects of TLR4 expression in chemoattrac-tion and activation of DC, we inoculated parental 3LL tumorcells in TLR4/knockout mice. We found that the tumor in-filtration of DC in TLR4-deficient mice was slightly but notsignificantly decreased before and after HT (data not shown).However, these tumor-infiltrating DC in TLR4-deficient micewere found to be impaired in their phenotypic maturation (Sup-plemental Fig. S9B) and chemokine receptor CCR7 and CXCR4expression (Supplemental Fig. S10B) promoted by HT. Corre-spondingly, the DC in spleen and lymph nodes of TLR4/

mice bearing parental 3LL tumor after HT were significantlydecreased (Fig. 7C).

Finally, to show the importance of releasable HSP70 in theinduction of specific antitumor immunity, we analyzed theCD8 CTL induction in the mice-bearing HSP70 (both HSC70and HSP70i)-silenced 3LL tumor. We found that the IFN- pro-duction and cytotoxicity of CD8 T cells were both decreased,as compared with that in the mice-bearing scrambled control3LL tumor (Fig. 7, D and E). These data further demonstratedthe important roles of releasable HSP70 in the induction ofspecific CD8 CTL.

DiscussionVarious HSP were released from tumor cells upon HS treatment(3–5). However, we found that HSP70 (including HSP70i andHSC70) may be the major HSP responsible for the autocrine in-duction of chemokines by tumor cells, as evidenced by our siRNAexperiments showing that HSP70 silence, but not HSP90, or gp96or HSP60 silence, could significantly impair the induction of che-mokines and the subsequent chemoattraction of DC by tumor cells.Moreover, the HSP70 (both HSC70 and HSP70i)-silenced 3LLtumor demonstrated less DC infiltration and impaired phenotypic/functional maturation of the tumor-infiltrating DC, indicating thatHT-induced infiltration of DC and other immune cell populationsmay be due to the tumor-released HSP70, and HSP70 released bytumor cells after HT may be prominent in the activation of infil-trated DC. We also have provided evidence that DC infiltratedwithin tumor after HT showed the elevated expression of CXCR4and CCR7, indicating that HT may induce DC maturation andpromote the migration of DC from tumor tissues to secondarylymphoid organs. Twenty-four hours after HT, DC in the spleenand lymph nodes were increased and showed the improved capac-ity in the induction of tumor Ag-specific CTL, which was con-firmed by our FACS assays of DC within spleen and lymph nodesand by our in vitro CTL induction assays. The major involvementof HSP70, but not HSP90 and HSP60, in HT-induced chemoat-traction and activation of DC may be due to the differential levelsof these HSP released by tumor cells and the different efficiency ofthese HSP in activating target cells (1, 2, 5, 12, 35). When HSC70was silenced alone, the chemokine production by 3LL cells afterHS was much lower than that of HSP70i-silenced 3LL cells,strongly suggesting that the levels of HSP released by tumor cellsmay significantly affect the relative contribution of each HSP toproduction of chemokines during HS (data not shown).

It has been reported that HSP90, HSP70, and gp96 derived fromtumor cells may contain tumor Ags and can present the associatedAgs to APC, finally resulting in Ag-specific CTL induction (6). Inour studies, we did not examine the HSP70-, HSP90- and gp96-chaperoned Ags released from tumor cells after HT, which sug-gested that these HSP may be capable of presenting tumor Ags toDC, given that releasable HSP70 can increase chemokine produc-tion by tumor cells and promote the infiltration, activation, andmigration of DC. Therefore, our studies have suggested a potentialmechanism for the initiation of antitumor immunity during the HT


treatments. HT may induce the release HSP70 (including HSC70and HSP70i) from tumor cells, which can activate tumor cells viaautocrine manner for chemokine production and simultaneouslyactivate the chemoattracted DC via paracrine manner, finally lead-ing to the uptake of tumor Ags chaperoned by HSP and the sub-sequent presentation of tumor Ags to naive T cells in spleen andlymph node once the DC mature and migrate to spleen and lymphnodes. Considering that local HT treatments were administeredrepeatedly, we suggest that the autocrine and paracrine actions ofreleasable HSP70 may be positively augmented, leading to theobserved inhibition of tumor growth and induction of antitumorimmunity. Whether or not other HSP, such as HSP90, gp96, andHSP60, are also involved in the induction of chemokines and ac-tivation of DC needs further investigation.

HSP can bind and activate target cells via HSP receptors. Manymolecules have been suggested as receptors for HSP, includingTLR2/4, CD91, CD40, CD14, and so on (25–34). In our study, wehave identified TLR4 as the major receptor for HSP70 in the in-duction of chemokines and activation of DC both in vitro and invivo. It has been suggested that HSP70-induced proinflammatorycytokine production is mediated via the MyD88/IL-1R-associatedkinase/NF- B signal transduction pathway and that HSP70 usesboth TLR2 and TLR4 to transduce its proinflammatory signal in aCD14-dependent fashion (25, 26, 29, 31). We found that silence ofCD91 and CD40 expression in 3LL cells didn’t affect the inductionof CXCL10 after HS treatments, whereas TLR4 and CD14 silencecould affect the chemokine induction. TLR4 silence almost elim-inated HS-induced activation of NF- B and IRF3 signaling path-ways, suggesting that TLR4 may play a major role in autocrineactivation of tumor cells by HSP70, and CD14 may help the TLR4signal transduction in a similar manner to those reported for LPSrecognition by macrophages (25, 27). One inconsistency of ourfindings is the exclusion of TLR2 in mediating the signaling by thereleased HSP70. However, we have shown that 3LL cells are neg-ative for TLR2 expression, and HS treatments can activate theTRIF-dependent activation of IRF3. Previous studies have sug-gested that activation of TLR2 induces the expression of CXCL8,CCL5, CCL3, and CCL4, whereas activation of TLR4 induces theexpression of CXCL10, CCL5, CCL3, and CCL4 (22, 36). There-fore, TLR4, but not TLR2, is responsible for the releasable HSP70-induced tumor cell production of chemokines in autocrine mannerafter HT treatment by activating both MyD88- and TRIF-depen-dent signaling pathways.

In sum, we have demonstrated the important roles of releasableHSP from heat-stressed tumor cells in the initiation of antitumorimmunity. Our studies suggest that HSP70 (including HSC70 andHSP70i) released from tumor cells after HT is the major factorresponsible for subsequent autocrine induction of chemokines andchemoattraction of DC and for the paracrine activation of DC viaTLR4 signaling pathways. We have also provided evidence for theimportance of TLR4 in the tumor immunotherapy. Our data sug-gest that TLR4, under certain conditions (e.g., HT), can favorablyelicit antitumor immunity through facilitating chemokine produc-tion by tumor cells and through promoting Ag presentation by DC.Thus, our study provides a potential explanation for the mecha-nisms involved in HT-induced antitumor immunity.

AcknowledgmentsWe appreciate Drs. Qiuyan Liu and Minghui Zhang for helpful discussionand Yan Li, Ting Zhang, and Mei Jin for their excellent technicalassistance.

DisclosuresThe authors have no financial conflict of interest.

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