ARTICLE IN PRESS

Immunobiology 215 (2010) 535–544

Contents lists available at ScienceDirect

Immunobiology

0171-29

doi:10.1

Abbre

nuclear

MHC, m

transcri� Corr

Center,

85724-5

E-m

katsanis1 Em

journal homepage: www.elsevier.de/imbio

Signaling pathways induced by a tumor-derived vaccine in antigenpresenting cells

Jessica Cantrell a,b, Claire Larmonier a,c, Nona Janikashvili a, Sara Bustamante a,b, Jennifer Fraszczak d,Amanda Herrell a, Tamara Lundeen a, Collin J LaCasse a,c, Elaine Situ a, Nicolas Larmonier a,b,c,e,�,1,Emmanuel Katsanis a,b,c,e,�,1

a Department of Pediatrics, Steele Children’s Research Center, University of Arizona, 1501 N. Campbell Ave., PO Box 245073, Tucson, AZ 85724-5073, USAb Cancer Biology Graduate, University of Arizona, 1501 N. Campbell Ave., PO Box 245073, Tucson, AZ 85724-5073, USAc Department of Immunobiology, University of Arizona, 1501 N. Campbell Ave., PO Box 245073, Tucson, AZ 85724-5073, USAd INSERM UMR 866, IFR 100, Faculty of Medicine, Dijon, Francee BIO5 Institute and Arizona Cancer Center, University of Arizona, 1501 N. Campbell Ave., PO Box 245073, Tucson, AZ 85724-5073, USA

a r t i c l e i n f o

Article history:

Received 1 July 2009

Received in revised form

13 September 2009

Accepted 16 September 2009

Keywords:

Tumor vaccines

Dendritic cells

Macrophages

Cell signaling

85/$ - see front matter & 2009 Elsevier GmbH

016/j.imbio.2009.09.006

viations: CRCL, chaperone-rich cell lysates;

factor kappa B; NK, natural killer cells; MAP,

ajor histocompatibility complex; STAT, signal

ption

esponding authors at: Department of Pediatri

University of Arizona, 1501 N. Campbell Ave.

073, USA. Tel.: +1520 626 4851; fax: +1520

ail addresses: nrlarmon@email.arizona.edu (N

@peds.arizona.edu (E. Katsanis).

manuel Katsanis and Nicolas Larmonier sha

a b s t r a c t

We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL)

vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent

immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides

both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans,

tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the

generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling

events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-

derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the

function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is

associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT

and with transcription factor NF-kB activation in vitro and in vivo. These results thus provide important

insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen

presenting cells.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

Heat shock proteins (HSP), also known as stress proteins orchaperone proteins, are the most abundant intracellular proteinspresent in all living organisms (Smith et al. 1998). They display amultitude of housekeeping functions, including the folding ofnascent polypeptides, refolding of denatured proteins and stabi-lizing proteins against aggregation and intracellular transport.Chaperone proteins are essential for cellular survival and forprotecting cells from a variety of damaging environments. Purified

. All rights reserved.

DC, dendritic cells; NF-kB,

mitogen-activated protein;

transduction and activator of

cs, Steele Children’s Research

, PO Box 245073, Tucson, AZ

626 6986.

. Larmonier),

re senior authorship.

individual tumor-derived chaperone proteins such as HSP 70, HSP90, GRP94/gp96, and calreticulin (CRT) have also shown promiseas vaccines which are capable of generating tumor-specific T cellresponses leading to protective anti-cancer immunity (Heike et al.1999; Li 1997; Srivastava and Amato 2001; Srivastava et al. 1998;Wells and Malkovsky 2000). The underlying principle for the useof chaperone proteins as vaccines is based on their intrinsicfunction as intracellular carriers of antigenic peptides. The captureof chaperone proteins and their associated antigenic peptides byantigen presenting cells (APC) may foster the delivery of thesepeptides into the antigen-processing pathways and may lead totheir MHC-dependent presentation to T cells (Li et al. 2002;Srivastava 2002a, 2002b). Some groups have reported thatchaperone proteins may also act as natural adjuvants or ‘dangersignals’ that are capable of activating antigen presenting cells(Segal et al. 2006; Srivastava et al. 1998). This effect has howeverbeen disputed by other studies suggesting that endotoxincontamination or LPS binding to chaperone proteins purified fromtissues may play a major role in APC activation (Gao and Tsan2003; Nicchitta 2003; Radsak et al. 2003; Reed et al. 2003; Wallinet al. 2002).

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544536

A cancer cell-based vaccine has been developed in ourlaboratory which consists of tumor lysates enriched for the majorimmunogenic chaperone proteins using a free solution-isoelectricfocusing technique (FS-IEF) (Graner et al. 2000a, 2004; Zenget al. 2006). The enriched, tumor-derived chaperone proteinsform large, multi-chaperone complexes termed chaperone-richcell lysate(s) (CRCL) that contain the HSP90 and HSP70 familymembers, the endoplasmic reticulum chaperones glucose regu-lated protein (GRP)94/glycoprotein (gp)96, and calreticulin(Graner et al. 2000a, 2000b, 2003; Zeng et al. 2006). CRCL is thusdifferent from conventional individually purified chaperonepreparation used as vaccine. In numerous animal models, CRCLdemonstrated a more pronounced anti-tumor effect per unitmaterial of protein than the individual purified chaperoneprotein alone (Graner et al. 2000a, 2000b, 2003; Zeng et al.2003). CRCL elicited specific T cell responses resulting in tumorregression (Graner et al. 2000a, 2000b, 2003). Previous resultshave indicated that the efficacy of this vaccine stems in part fromits ability to induce dendritic cell (DC) activation through up-regulation of CD40 expression and IL-12 production (Zeng et al.2003).

The objective of this study was to identify the main signalingevents and modifications associated with CRCL-induced activationof APC. We herein demonstrate that tumor-derived CRCL, inaddition to triggering DC pro-inflammatory cytokine secretionalso promotes the up-regulation of the recently described co-stimulatory molecule, CD70. In addition, CRCL stimulates thedirect tumor killing activity of macrophages, which is associatedwith inducible nitric oxide synthase (iNOS) expression and nitricoxide (NO) production. The molecular signaling events associatedwith CRCL-mediated activation of APC involve the induction of theMAP kinase pathway evidenced by increased phosphorylation ofERK1/2 and p38, the activation of the transcription factor NF-kBand the phosphorylation of STAT1, STAT5 and AKT.

Materials and methods

Mice

Mice were housed under specific pathogen-free conditions andcared for according to the guidelines of the University of ArizonaInstitutional Animal Care and Use Committee. Female BALB/c(H2d) mice were obtained from the National Cancer Institute(Bethesda, MD) and used at the age of 6–8 weeks.

Cell lines

The murine leukemia cell line 12B1 (given by Dr. Wei Chen(Cleveland Clinic, Cleveland, OH) was obtained by retroviraltransformation of BALB/c mouse bone marrow cells with thehuman bcr/abl (b3a2) fusion gene and express the p210 bcr-ablprotein (McLaughlin et al. 1987). This is an aggressive leukemia,with the 100% lethal dose (LD100) being 100 cells after tail veininjection (He et al. 2001). The murine macrophage cell lineRAW264.7 and the melanoma tumor cell line B16 were purchasedfrom the American Type Culture Collection (Manassas, Virginia).Cells were cultured at 37 1C and 10% CO2 in DMEM or RPMI media(Gibco/BRL, Gaithersburg, MD) containing 10% heat-inactivatedfetal calf serum (Gemini Bio-products, Woodland, CA) andsupplemented with 2 mM glutamine, 100 U/ml penicillin, 100mg/mlstreptomycin sulfate, 0.025mg/ml amphotericin B, 0.5� MEMnonessential amino acids and 1 mM sodium pyruvate (completemedia, CM). Cells were tested routinely and found to be free ofMycoplasma contamination.

Bone marrow-derived DC and peritoneal macrophages

DC were generated from BALB/c bone marrow cells aspreviously reported (Larmonier et al. 2008b). Cells were harvestedfrom femurs and tibiae and filtered through a Falcon 100-mmnylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ).Red blood cells were lysed in a hypotonic buffer (150 mM NH4Cl,1 mM KHCO3, 0.1 mM Na2EDTA) and total bone marrow cells werecultured at a density of 5�105 cells/ml in a complete RPMImedium. Murine granulocyte-macrophage colony-stimulatingfactor (GM-CSF; Peprotech, Rocky Hill, NJ) and Interleukin-4 (IL-4; Peprotech) were added at the concentration of 10 ng/ml each.Complete RPMI medium containing GM-CSF and IL-4 was addedon day 2. On day 5 non-adherent and loosely adherent cells werecollected, washed in complete RPMI and used in further experi-ments. Flow cytometry analysis indicated that approximately 70%of the cells expressed CD11c. Macrophages were obtained bywashing BALB/c naıve mouse peritoneal cavity with 5 ml of coldRPMI medium. Macrophage number was determined by countingadherent cells after a 1-h attachment period on a hemocytometerglass surface at 37 1C in 5% CO2. Cells were seeded and used after a24 h adherent step in a flat bottom 96-well plate followingelimination of floating cells by extensive washes.

CRCL preparation

Tumor-derived CRCL (12B1 or B16) was prepared as describedpreviously (Graner et al. 2000a, 2000b, 2003). The endotoxin levelcontained in the CRCL preparation was determined using theLimulus Amebocyte Lysate (LAL) assay kit (Cambrex Bio Science,Walkersville, MD) according to the manufacturer’s instructions.The level of endotoxin in CRCL was lower than that in mediacontrol (o0.01 EU/mg).

Detection of cytokine and chemokine production by ELISA

The concentration of TNF-a, IL-12 or RANTES in DC ormacrophage culture supernatants was determined using en-zyme-linked immunosorbent assay (ELISA) kits according to themanufacturer’s instructions (eBiosciences, San Diego, CA).

Real-time PCR

Total RNA was extracted using TRIzol reagent (GibcoBRL). Tenmicrograms of total RNA was treated with DNase I according tothe manufacturer’s protocol (Ambion, Austin, TX). The resultingRNA was evaluated by agarose gel electrophoresis and concentra-tions determined by spectrophotometry on an MBA 2000 spectro-photometer (Perkin Elmer, Boston, MA). 500 ng of DNase I-treatedRNA was reverse-transcribed using Bio-Rad iScript (Bio-Rad,Hercules, CA) according to the manufacturer’s protocol. Subse-quently, 20-ml PCR reactions were set up in 96-well platescontaining 10ml of TaqMan universal PCR master mix, 1ml ofTaqMan primer/probe set, 2ml of cDNA synthesis reaction (out of a20-ml total volume), and 7ml of molecular grade water. Reactionswere run and analyzed on a Bio-Rad iCycler iQ real-time PCRdetection system. Data were analyzed using the comparative Ctmethod as a means of relative quantization, normalized toan endogenous reference (18S ribosomal RNA) and relative to acalibrator (normalized CT value obtained from medium-alonecells) and expressed as 2–DCT according to Applied BiosystemsUser Bulletin 2: Rev B Relative Quantitation of Gene Expression.The real-time PCR products were separated on a 2% agarose E-Gel(Invitrogen, Carlsbad, CA) and visualized by ethidum bromide.

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544 537

Flow cytometry analysis and antibodies

Day 5 DC cultured as described above and treated or not for48 h with LPS or tumor-derived CRCL, with or without anti-CD40Abs were incubated for 10 min with an Fc receptor-blockingantibody (BD Biosciences Pharmingen, San Diego, CA, USA), thenfor 30 min with saturating amounts of the appropriate primaryantibodies in PBS. Cells were then washed three times in PBSand analyzed using a FACScan (Becton Dickinson Immunocyto-metry Systems, San Jose, CA, USA). A minimum of 10,000 eventswere recorded for each sample and data were analyzed using aCellQwest software (Becton Dickinson Immunocytometry Sys-tems). The following antibodies were used: PE-anti-CD70(eBioscience, San Diego, CA, USA) and APC-anti-CD11c (MiltenyiBiotech). Isotype control antibodies were obtained from BDBiosciences Pharmingen.

Detection of NF-kB and iNOS by immunohistochemistry

Mice were injected (s.c) with PBS, LPS (1mg/ml) or 12B1 tumor-derived CRCL (25mg/ml). After 24 h, draining lymph nodes wereremoved, embedded in Tissue-Tek (Sakura Finetek USA, Torrance,CA) or snap-frozen in liquid nitrogen. Cryostat tissue sectionswere prepared and slides were treated as described (Larmonieret al. 2008a). RAW264.7 macrophages were plated on slidechambers, exposed for 24 h to tumor-derived CRCL (25mg/ml) orLPS (1mg/ml), fixed with methanol and permeabilized with 0.1%saponin. Slides were incubated with a primary antibody againstiNOS or phospho-NF-kB p65 (RelA) (Ser276; 1/50 dilution, CellSignaling Technology, Danvers, MA) in PBS containing 1% BSA,overnight at 4 1C. After three washes in PBS, slides were incubatedwith biotinylated secondary antibody and streptavidin/peroxidasecomplex according to the manufacturer’s recommendation (KPL).Slides were then incubated with 3,3’-diaminobenzidine (Vectorlaboratories, Burlingame, CA) and mounted with Vectamountmedium (Vector Laboratories). Slide examination was performedindependently by two experienced scientists in a blind mannerusing a Zeiss Axioplan microscope (Carl Zeiss MicroImaging,Thornwood, NY). Images were captured with Nikon Digital SightDS-Fi1 camera and NIS-Element software (Nikon Instruments,Melville, NY). Staining was scored by counting the number ofpositive cells per high-power field (200� magnification). A totalof six fields in three randomly chosen sections were analyzed foreach group.

Detection of the expression of iNOS and of the phosphorylation of

I-kB, STAT1, STAT3, STAT5, ERK1/2, p38 and AKT by western blotting

DC and peritoneal macrophages were cultured as describedabove. RAW264.7 cells were cultured in 25 cm2 flasks. Cells werelysed in a lysis buffer (1% Nonidet P40, 50 mM Tris–HCl, pH 7.4,2 mM EDTA, 100mM NaCl, 0.2 mg/mL Aprotinin, 0.2 mg/mLLeupeptin, 1 mM PMSF, 10 mM NaF, 30 mM NaPPi, 10 mM Na3VO4).DC or macrophages treated with LPS were used as positive control.Negative controls consisted of DC or macrophages culturedalone. Western blot analysis was then performed as described(Larmonier et al. 2007), using the following antibodies: anti-phospho I-kB, anti I-kB, anti-phospho STAT1, anti-STAT1, anti-phospho STAT3, anti-STAT3, anti-phospho STAT5, anti-STAT5,anti-phospho ERK1/2, anti-ERK1/2, anti-phospho p38, anti-p38,anti-phospho AKT, anti-AKT, anti-phospho IRAK1 or anti-IRAK1(Cell Signaling, Beverly, MA) or anti-iNOS (Santa Cruz Biotechnol-ogy, Inc., Santa Cruz, CA), or anti-b-actin (Sigma, St. Louis, MO).

Determination of nitrite production

The amount of NO2� released by macrophages was detected in

fresh cell-free supernatants by the colorimetric Griess reagent kitas described by the manufacturer’s instructions (MolecularProbes, Eugene, OR). The results were expressed in micromolesof NO2

� per ml.

Evaluation of macrophage killing activity

Peritoneal macrophages obtained as described above andtreated with LPS (1mg/ml) or CRCL (25mg/ml) were cultured withtumor cells for 24 h. Tumor cell killing was assessed using crystalviolet assays as reported (Larmonier et al. 2006). The death ofdetached tumor cells was confirmed by trypan blue staining andby the inability of these cells to form colonies when sub-culturedin fresh culture medium for 4 days.

Detection of NF-kB activation by DNA-binding transcription factor

ELISA assay

Nuclear extracts were harvested from DC (1�106 cells) ormacrophages (5�105 cells) using a nuclear extract kit (ActiveMotif, Carlsbad, CA). NF-kB P50 DNA-binding activity was thenassessed using 15mg of nuclear extracts according to themanufacturer’s recommendations (NF-kB P50 Trans-AM

TM

kit,Active Motif). DC or macrophages treated with LPS (1mg/ml) wereused as positive control.

NF-kB plasmid transfection and luciferase assay

Cells were transiently transfected using the TransIT expresstransfection reagent according to manufacturer’s instructions(Mirus Bio, Madison, WI). After cell lysis, reporter gene assaywas performed according to manufacturer’s instruction (Lucifer-ase Reporter assay system; Promega, Madison, WI). Luciferaseactivity was measured with a tube luminometer (FemtomasterFB12, Berthold Detection System, Pforzheim, Germany) andexpressed as normalized luciferase activity relative to proteinconcentration (BCA assay; Pierce, Rockford, IL).

Statistical analysis

Unless specified otherwise, all experiments were performed intriplicate and reproduced three times. Statistical analyses compar-ing values between groups were performed using the Student’st-test or by the ANOVA followed by Fisher paired least-significantdifference (PLSD) post hoc test with StatView software packagev.4.53 (SAS Institute, Cary, NC).

Results

Tumor-derived CRCL induces CD70 expression by DC

CRCL was produced from 12B1 leukemia tumors as previouslyreported (Graner et al. 2000a). Before analyzing the signalingevents and changes induced by CRCL in APC, it was critical toensure that this particular vaccine preparation was biologicallyactive. Consistent with our previous results (Larmonier et al.2007; Zeng et al. 2003), tumor-derived CRCL enhanced pro-inflammatory cytokine and chemokine production by DC andprimary macrophages (Supplemental figure S1) and by the murinemacrophage cell line RAW264.7 (Supplemental figure S2).

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544538

We have previously reported that CRCL up-regulates theexpression of co-stimulatory molecules CD40 and to a lesserextent CD80 and CD86 in DC (Zeng et al. 2003). CD70 is a recentlydescribed co-stimulatory molecule expressed by mature DC. Theinteraction of CD70 with its ligand CD27, expressed by resting Tcells, activated B cells, and NK cells, is critical for the developmentof potent cell-mediated immunity (Tesselaar et al. 2003). It hasbeen suggested that some TLR ligands such as Pam3Cys-SK4(TLR1/2 agonist), Poly(I:C) (TLR3 agonist), and MPL (TLR4 agonist)combined with CD40 signaling may promote optimal expressionof CD70 (Sanchez et al. 2007). Based on these observations, wereasoned that tumor-derived CRCL may induce up-regulationof DC CD70 expression. To test this hypothesis bone marrow-derived DC were treated with LPS or 12B1 tumor-derived CRCL,in the presence or absence of an anti-CD40 antibody. Expressionof CD70 was then determined by flow cytometry. The resultsdepicted in Fig. 1A indicate that CRCL was capable of inducingthe up-regulation of CD70 by DC. Furthermore, CD70 expressionwas significantly enhanced by the presence of anti-CD40antibodies (Fig. 1B and C). These results thus highlight CD70 as

0

5

10

15

20

25

30

Anti-CD40 LPSAn

Mea

n pe

rcen

t inc

reas

e

CD

11c

CD7

CD7

CD

11c

5.71%

11.69%

Untreated

Anti-CD40LPS

anti CD

LP

Fig. 1. CRCL induces the up-regulation of the co-stimulatory molecule CD70 by DC. (A) D

LPS (1mg/ml) as positive control. (B) Similar as (A) but in presence of anti-CD40 antibod

PE-conjugated anti-CD70 mAb and analyzed by flow cytometry. (A, B) Representative

expression by DC following the indicated treatments compared to untreated DC. *, a si

an additional co-stimulatory molecule induced by tumor-derivedCRCL.

CRCL enhances macrophage iNOS expression, NO production and

tumoricidal activity

Macrophages represent a population of cells located at thenexus between innate and adaptive immunity. They may act asantigen presenting cells as well as effector killer cells and havebeen reported to be involved in the regulation of tumor immunity(Bonnotte et al. 2001; Klimp et al. 2002; Kryczek et al. 2006).Based on the central role of these cells in tumor growth controland on our observation that tumor-derived CRCL prominentlypromote macrophage activation, we sought to further definewhich aspect(s) of macrophage function(s) may be modulatedby the CRCL vaccine. Activated macrophages up-regulate iNOSexpression, the inducible form of nitric oxide synthase, whichleads to nitric oxide (NO) production (Bogdan 2001). NO is a majoreffector molecule involved in macrophage cytotoxic activity

LPS +ti-CD40

CRCL +Anti-CD40

CRCL

*

0

0

20.54% 15.21%

22.98% 27.84%

+40

CRCL + anti-CD40

S CRCL

ay 5 bone marrow derived DC were treated with tumor-derived CRCL (25mg/ml), or

y (5mg/ml). After 48 h cells were stained with APC-conjugated anti-CD11c mAb and

dot plots of three independent experiments. (C) Mean percent increase in CD70

gnificant difference compared to DC treated with CRCL alone (po0.05).

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544 539

(Bogdan 2001; Klimp et al. 2002). We therefore sought todetermine whether CRCL may stimulate iNOS expression andNO secretion. IHC staining of the RAW264.7 macrophage cell lineindicates that the number of iNOS-expressing cells was signifi-cantly increased following CRCL (975 iNOS positive cells/field,po0.01) or LPS (1674 iNOS positive cells/fields, po0.001)treatment compared to untreated cells (0.570.8 iNOS positivecells/fields) (Fig. 2A). Western blot analysis further demonstratesthat CRCL was capable of triggering iNOS expression in RAW264.7and primary (peritoneal) macrophages (Fig. 2B). Furthermore, theconcentration of nitrites, the principal metabolites of NO, wasincreased in the culture supernatants of CRCL-activated peritonealor RAW264.7 macrophages (Fig. 2C). LPS and CRCL induced theproduction of comparable levels of nitrites, but the expressionof iNOS was higher in CRCL-treated RAW264.7 macrophagessuggesting a reduced activity of this enzyme. We next evaluatedthe influence of CRCL on the direct killing activity of macrophagestowards cancer cells. The results depicted in Fig. 3 demonst-rate that CRCL significantly promoted the cytotoxic potential ofmacrophages against tumor cells (Fig. 3). No direct effects of LPSor CRCL on cancer cells were observed (not shown). The death of

Untreated LPS

1

2

3

4

5

6

Nitr

ite C

once

ntra

tion

(μM

)

β-actin

iNOS

β-actin

iNOS

RAW264.7

Peritoneal Mφ

Fig. 2. CRCL induces iNOS expression and NO production by macrophages. (A) RAW264

(25mg/ml). Cells were then washed and stained for iNOS expression by IHC. Upper p

detection of iNOS expression in RAW264.7 or peritoneal macrophage treated with CRCL

with CRCL (25mg/ml) and nitrite concentration was evaluated in the culture supernatant

difference when compared to untreated control cells (po0.05).

detached tumor cells upon contact with macrophages wasconfirmed by trypan blue staining (9775% trypan blue positivedead cancer cells for the CRCL-treated macrophage group and9477% for the LPS-treated macrophage group) and the inabilityof these detached cells to form colonies when sub-cultured infresh culture medium for 4 days (not shown). Taken together,these findings demonstrate that CRCL stimulates the tumoricidalfunction of macrophages which is associated with the induction ofiNOS expression and NO production and further highlight thesecells as major targets of CRCL.

CRCL triggers NF-kB activation in DC and macrophages

The transcription factor NF-kB plays a key role in the activationof DC and macrophages (Kawai and Akira 2006). Its activationresults in the secretion of pro-inflammatory cytokines andchemokines and is associated with NO production (Binder et al.2004). NF-kB activation depends on the cytoplasmic phosphor-ylation of its inhibitor I-kB by I-kB kinase (Akira and Takeda 2004;Kawai and Akira 2006). Once phosphorylated, I-kB dissociates

CRCL

0

0

0

0

0

0

0

012345678

RAW264.7

Peritoneal Mφ

untreated LPS CRCL

* *

*

*

.7 cells were plated on slide chambers and exposed for 24 h to tumor-derived CRCL

anels, 200� magnification, lower panels 400� magnification. (B) Western blot

(25mg/ml). (C) RAW264.7 cells or peritoneal macrophages were incubated for 12 h

s. In all the experiments LPS (1mg/ml) was used as a positive control. *, a significant

ARTICLE IN PRESS

0

20

40

60

80

100

120

Medium + mφ + mφ+ LPS

+ mφ+ CRCL

Surv

ival

(%)

**

*

Tumor cells

Fig. 3. CRCL triggers macrophage tumoricidal activity. Peritoneal macrophages

(50,000 cells per well) were cultured with B16 tumor cells (20,000 cells per well)

and treated or not with tumor-derived CRCL (25mg/ml) or with LPS (1mg/ml). After

24 h cell viability was determined using a crystal violet toxicity assay. B16 cells

alone (medium) and B16 cells cultured with macrophages (+mj) were used as

negative controls. The data are representative of three independent experiments. *,

a significant difference when compared to tumor cells cultured alone (po0.05).

Peritoneal Mϕϕ

IκB

P-IκB

DC

00.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9untreated LPS CRCL

NF-

κB p

50 a

ctiv

atio

n (O

D 4

50nm

)

0

0.2

0.4

0.6

0.8

1

1.2

Macrophages

DC

35

*

*

*

*

untreated LPS CRCL

J. Cantrell et al. / Immunobiology 215 (2010) 535–544540

from NF-kB that translocates into the nucleus where it binds to itsspecific DNA promoter sequence upstream of target genes. Theresults depicted in Fig. 4A indicate that tumor-derived CRCL iscapable of triggering I-kB phosphorylation in both bone marrow-derived DC and peritoneal macrophages. Compared to LPS,CRCL induced a higher phosphorylation of I-kB in DC than inmacrophages. To confirm these results, we performed DNA-binding transcription factor ELISA to detect NF-kB activationin CRCL-treated DC and macrophages. NF-kB p50 binding toits specific oligonucleotide probe was enhanced in the nuclearextracts prepared from CRCL-treated cells (Fig. 4B). CRCL-inducedNF-kB activation was further confirmed using a NF-kB luciferasereporter gene introduced in the RAW264.7 macrophage cellline. Our results indicate that CRCL treatment results in anincrease in NF-kB activation when compared to untreated cells(Fig. 4C).

To determine whether our in vitro findings translate in vivo, weinvestigated the phosphorylation of NF-kB p65 in the draininglymph nodes (DLN) of naıve mice injected with PBS, LPS or CRCL.We detected a significant increase in NF-kB p65 phosphorylationin the DLN of animals administered CRCL vaccine when comparedto control (PBS-treated) mice (Fig. 5). These results thereforedemonstrate that CRCL induces activation of NF-kB both in vitro

and in vivo.

Fig. 4. Activation of NF-kB in CRCL treated DC and macrophages. (A) Day 5 bone

marrow-derived DC or peritoneal macrophages were incubated for 24 h with CRCL

(25mg/ml). Total cell extracts were performed and phospho-IkB or IkB expression

was analyzed by Western blot. Untreated cells were used as a negative control and

cells treated with LPS (1mg/ml) were used as a positive control. (B) Day 5 bone

marrow-derived DC or peritoneal macrophages were incubated for 24 h with CRCL.

Nuclear extracts were performed and the DNA-binding activity of NF-kB p50 to a

consensus DNA probe was assessed as described in Materials and methods. DC or

macrophages cultured alone were used as a negative control, and cells treated with

LPS were used as a positive control. The data are shown as a mean7SD of

duplicate wells of NF-kB p50 activation determined as the OD value (450 nm) as

indicated by the manufacturer. (C) RAW264.7 cells were transiently transfected

with a NF-kB luciferase plasmid. After 36 h, cells were treated with LPS or CRCL for

12 h as indicated and a luciferase assay was performed as described in Material

and methods. Data are presented as relative luminescent unit (RLU)/mg of protein.

Results are representative of three independent experiments. *, a significant

difference when compared to untreated control cells (po0.05).

CRCL induces the activation of STAT1, STAT5, AKT and the MAP kinase

pathway in DC and macrophages

To further explore the mechanisms by which CRCL activates DCand macrophages we investigated the role of additional transcrip-tion factors and upstream components of the NF-kB signalingcascade.

RLU

/μg

X 10

4

0

5

10

15

20

25

30RAW264.4

**

ARTICLE IN PRESS

PBS LPS CRCL

Num

ber o

f pho

spho

-NF-

κκBp6

5po

sitiv

e ce

lls/fi

eld

PBS LPS CRCL

0

2

4

6

8

10

12

14

16*

Fig. 5. CRCL-induced phosphorylation of NF-kB p65 in vivo. Naıve mice (4 animals per group) were injected (s.c.) with CRCL (100ml, 25mg), LPS (100ml, 1mg) or PBS as

control vehicle. Phospho-NF-kBp65 was detected in the draining lymph nodes by IHC staining. Upper panels, 200� magnification, lower panels 400� magnification.

Histogram represents a double blind count of 10 frames of phospho-NF-kB p65 positive cells. *, a significant difference compared to untreated mice (po0.002).

J. Cantrell et al. / Immunobiology 215 (2010) 535–544 541

STAT1, a transcription factor activated following phosphoryla-tion, has been shown to promote DC and macrophage activation(Shuai and Liu 2003). Compared to untreated cells, an increase ofSTAT1 Tyr701 phosphorylation was detected in DC and macro-phages treated with CRCL (Fig. 6 and Supplemental Figure S3).STAT5 is an additional transcription factor activated in responseto a variety of cytokines and growth factors and is also involvedin the activation of DC and macrophages (Hennighausen andRobinson 2008). The phosphorylation of STAT5 Tyr694 wasenhanced in DC and macrophages treated with CRCL (Fig. 6 andSupplemental Figure S3). Conversely, CRCL reduced Tyr694

phosphorylation of STAT3, a transcription factor that dampensDC and macrophage activation (Cheng et al. 2003) (Fig. 6).

The MAP kinase (MAPK) pathway is activated in responseto various cell surface signals that converge to the activation oftranscription factors such as NF-kB or STAT-1 and STAT-5 (Akira andTakeda 2004). To further identify the signaling cascades triggered inresponse to CRCL in DC and macrophages, we evaluated a possiblemodifications of MAPK components. Our results indicate that thephosphorylation of ERK1/2 Thr202/Tyr204 and p38 Thr180/Tyr182,critical components of the MAP kinase cascade was increased inCRCL-treated DC and macrophages. The phosphorylation of ERK1/2induced by LPS or CRCL was higher in macrophages compared to DC(Fig. 6 and Supplemental Figure S3).

AKT represents an important signaling protein involved incellular survival pathways. It regulates apoptotic processes andmodulates downstream effector molecules such as NF-kB (Akiraand Takeda 2004). The results depicted in Fig. 6 demonstrate thatCRCL promotes the phophorylation of AKT Ser473 in DC and to ahigher extend in macrophages. These data thus indicates that

CRCL is capable of inducing cell survival pathways within antigenpresenting cells, particularly in macrophages.

CRCL overcomes TGF-b-mediated inhibition of DC

We have previously reported that a subset of immunosup-pressive lymphocytes (CD4+CD25+FoxP3+ regulatory T cells, Treg)suppress the induction of anti-tumoral immune responses byimpairing the activation of DC. This inhibition partly involvesthe immunosuppressive cytokine TGF-b (Larmonier et al. 2007).We have also reported that Treg fail to inhibit DC phenotypicmaturation or pro-inflammatory cytokine production followingexposure to tumor-derived CRCL (Larmonier et al. 2008a). Tofurther evaluate whether CRCL can overcome the inhibitory affectsof TGF-b on DC, these cells were treated with LPS or tumor-derived CRCL with or without TGF-b and TNF-a secretion wasdetermined. Our results indicate that DC stimulated with CRCLresist TGF-b-mediated suppression (Fig. 7).

Discussion

Chaperone proteins purified from tumor tissues have beenidentified as unique mediators of specific anti-cancer immunity.The advantages of using chaperone protein-associated peptides asa source of tumor antigens are multiple: (1) they do not requirethe identification of tumor-specific peptides, (2) they elicitpolyclonal T lymphocyte responses following immunization,preventing the outgrowth of immunological escape variants, (3)they consist of both MHC Class I associated peptides and MHC

ARTICLE IN PRESS

STAT1

P-STAT1

DC

STAT3

P-STAT3

P-p38

p38

P-ERK1/2

ERK1/2

P-AKT

AKT

P-STAT5

STAT5

Peritoneal Mϕ

Fig. 6. CRCL activates STAT1, STAT5, AKT and the MAP kinase pathway but impairs

the phosphorylation of STAT3 in DC and macrophages. Day 5 bone marrow-derived

DC or peritoneal macrophages were incubated for 24 h with CRCL (25mg/ml) or LPS

(1mg/ml) as a positive control. (A) Western blot analysis of total cell extracts was

then performed probing for phospho STAT1 (P-STAT1), STAT1, phospho STAT5 (P-

STAT5), STAT5, phospho ERK1/2 (P-ERK1/2), ERK1/2, phospho p38 (P-p38), p38,

phospho AKT (P-AKT), AKT, phospho STAT3 (P-STAT3), or STAT3.

TNF-

αα (p

g/m

l)

0

5000

10000

15000

20000

25000

untreated LPS CRCL

No TGF-β+ TGF-β

*

NS

Fig. 7. CRCL overcomes TGF-b-mediated DC suppression. Day 5 bone marrow-

derived DC were incubated for 24 h with CRCL (25mg/ml) or LPS (1mg/ml), with or

without TGF-b (10 ng/ml). The supernatants were harvested after 24 h and tested

for TNF-a production by ELISA. *, a significant difference when compared to DC

treated with LPS without TGF-b (po0.05). NS, non-significant difference when

compated to DC treated with CRCL without TGF-b.

J. Cantrell et al. / Immunobiology 215 (2010) 535–544542

Class II or helper epitopes, which together induce more potent anddurable immune responses, and (4) these proteins also act asnatural adjuvants or ‘danger signals’ capable of activating antigenpresenting cells such as DC and macrophages, enhancing thepotential of these cells to process and present antigens and tostimulate T cell responses (Asea et al. 2000; Basu et al. 2001;Binder et al. 2000; Srivastava and Udono 1994).

Our laboratory has developed an original tumor-derivedvaccine, CRCL, capable of eliciting specific T cell responses leadingto tumor regression (Graner et al. 2000a, 2000b, 2003). Theprimary components of CRCL are a combination of chaperones,which include HSP90, HSP70 family members, the endoplasmicreticulum chaperones glucose regulated protein (GRP)94/glyco-protein (gp)96, and calreticulin (Graner et al. 2000a, 2000b, 2003;Zeng et al. 2006). Each of these chaperones has been utilizedindividually and found to generate specific immunity against theirtumor of origin (Graner et al. 2000b; Srivastava 2002a). All of thesechaperones are gathered into a single preparation by the clusteringof the proteins that occur during the FS-IEF procedure (Graneret al. 2000a). While there are a number of other unidentifiedproteins in CRCL, immunodepletion of HSP70, HSP90, GRP94, andcalreticulin results in a significantly reduced immune response(Zeng et al. 2003, 2006), indicating that these chaperones are trulyresponsible for vaccine efficiency. In numerous animal models,CRCL vaccines have a more pronounced anti-tumor effect per unitmaterial of protein than any of the individual purified chaperoneproteins used alone or tumor lysate (Graner et al. 2000a, 2000b,2003; Zeng et al. 2003). Furthermore, the immunogenic potentialof CRCL can be enhanced by loading them into DC. The enhancedimmunogenicity of CRCL-pulsed DC may be due to the broadvariety of antigenic peptides that are chaperoned by CRCL, but alsoto the direct activation of DC by virtue of the adjuvant properties ofthe chaperone proteins within CRCL vaccines (Graner et al. 2003;Zeng et al. 2003, 2005).

Our previous studies have indicated that tumor-derived CRCLtriggers DC expression of CD40 and, to a lesser extent, CD80 and86, all of which are co-stimulatory molecules that are funda-mental for T cell activation. Our current study demonstrates thatCRCL additionally promotes the expression of CD70, a newlyidentified marker of activated DC that binds to CD27 expressed onnaıve T cells (Tesselaar et al. 2003). The engagement of CD27 withCD70 leads to sustained potent cellular immunity. The optimalup-regulation of CD70 involves both CD40/CD40L interaction aswell as stimulation through TLRs (Sanchez et al. 2007). Im-portantly, tumor-derived CRCL was as efficient as LPS in inducingCD70 up-regulation in DC, which highlights its effectiveness andfurther advocates for the advantages of using CRCL as a vaccinewith robust adjuvant qualities.

Macrophages represent a subset of immune cells capableof recognizing and killing cancer cells (Bauer et al. 2001; Bogdan2001; Nathan 1992; Pervin et al. 2001; Xie et al. 1996).Our previous studies had primarily focused on the modulationof dendritic cells by CRCL, but the effects of this vaccine onmacrophage functions were largely undetermined. We hereindemonstrate that macrophages represent prominent targets ofCRCL. Indeed, tumor-derived CRCL is not only capable of triggeringmacrophage pro-inflammatory cytokine production, but alsoinduces their tumoricidal activity. Macrophages can kill tumorcells by direct and/or indirect mechanisms including the produc-tion of nitric oxide (Bogdan 2001). Treatment with tumor-derivedCRCL results in the induction of macrophage iNOS expression andis associated with NO production. Furthermore, CRCL triggerssecretion of macrophage TNF-a that together with NO mayparticipate in the observed anti-tumoral activity. In addition,CRCL-activated macrophages may indirectly contribute to theinduction and maintenance of anti-tumor immune responses by

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544 543

secreting IL-12 and RANTES, which fosters DC activation and T cellrecruitment. These results thus demonstrate that by targeting notonly DC but also tumor killer macrophages, CRCL is endowed withthe potential to activate multiple arms of the anti-tumoralimmune responses, thus augmenting the diversity of the killingmechanisms leading to tumor elimination.

The molecular mechanisms underlying CRCL adjuvant effectson DC and macrophages had not been previously defined. Toaddress this question, we evaluated key signaling events that maybe modulated by CRCL in these cells. NF-kB is one of the keyfactors responsible for the transcription of genes involved in DCphenotypic and functional changes during activation (Iwasaki andMedzhitov 2004). NF-kB controls pro-inflammatory responses byup-regulating MHC Class II and co-stimulatory molecule expres-sion, as well as inducing the secretion of pro-inflammatorycytokines and chemokines by DC and macrophages (Akira andTakeda 2004; Kawai and Akira 2006). We here provide evidencethat the tumor-derived CRCL vaccine is capable of activatingNF-kB, both in vitro in DC and macrophages and in vivo followinginjection of mice. Further investigation led to the identification ofSTAT1 and STAT5 as additional transcription factors activated inDC and macrophages in response to CRCL stimulation. Further-more, CRCL activates the MAP kinase pathway, responsible forcell differentiation, survival, and growth (Dhillon et al. 2007).Additionally, CRCL may foster APC survival by promoting the Akt/PKB pathway. Interestingly CRCL hampers the phosphorylation ofSTAT3, a transcription factor that dampens the immune responseby inhibiting DC and macrophage activation and function (Shuaiand Liu 2003). Together these data demonstrate that tumor-derived CRCL promotes the intracellular signaling cascadesleading to DC and macrophage activation while hamperingnegative signals that may lead to their inhibition.

We have shown that regulatory T cells, a critical population oflymphocytes that contribute to tumor-induced immunosuppres-sion, inhibit DC activation though a mechanism involving TGF-b(Larmonier et al. 2007). However, this suppression of DC andmacrophages by regulatory T cells may overcome by the tumor-derived CRCL vaccine (Larmonier et al. 2008a). Consistent withthese data, we have now demonstrated that CRCL overrides TGF-bnegative signaling induced in bone marrow-derived DC. Theseresults therefore suggest that the positive cell signaling cascadesinduced by CRCL in DC may overcome the negative signalstriggered by tumor-induced suppressive factors or cells. Under-standing the mechanisms of action of this vaccine including itsadjuvant effects is critical in optimizing its use in future clinicaltrials.

Acknowledgements

This work was supported in part by the NIH grant R01CA104926 (EK), the Leukemia and Lymphoma Society FellowAward 5188-07 (NL), the Tee Up for Tots and Raise a Racquet forKids Funds (EK, NL).

Appendix. Supplementary materials

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.imbio.2009.09.006.

References

Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4,499–511.

Asea, A., Kraeft, S.K., Kurt-Jones, E.A., Stevenson, M.A., Chen, L.B., Finberg, R.W., Koo,G.C., Calderwood, S.K., 2000. HSP70 stimulates cytokine production through aCD14-dependant pathway, demonstrating its dual role as a chaperone andcytokine. Nat. Med. 6, 435–442.

Basu, S., Binder, R.J., Ramalingam, T., Srivastava, P.K., 2001. CD91 is a commonreceptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin.Immunity 14, 303–313.

Bauer, P.M., Buga, G.M., Fukuto, J.M., Pegg, A.E., Ignarro, L.J., 2001. Nitric oxideinhibits ornithine decarboxylase via S-nitrosylation of cysteine 360 in theactive site of the enzyme. J. Biol. Chem. 276, 34458–34464.

Binder, R.J., Han, D.K., Srivastava, P.K., 2000. CD91: a receptor for heat shock proteingp96. Nat. Immunol. 1, 151–155.

Binder, R.J., Vatner, R., Srivastava, P., 2004. The heat-shock protein receptors: someanswers and more questions. Tissue Antigens 64, 442–451.

Bogdan, C., 2001. Nitric oxide and the immune response. Nat. Immunol. 2,907–916.

Bonnotte, B., Larmonier, N., Favre, N., Fromentin, A., Moutet, M., Martin, M.,Gurbuxani, S., Solary, E., Chauffert, B., Martin, F., 2001. Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in arat model system. J. Immunol. 167, 5077–5083.

Cheng, F., Wang, H.W., Cuenca, A., Huang, M., Ghansah, T., Brayer, J., Kerr, W.G.,Takeda, K., Akira, S., Schoenberger, S.P., Yu, H., Jove, R., Sotomayor, E.M., 2003. Acritical role for Stat3 signaling in immune tolerance. Immunity 19, 425–436.

Dhillon, A.S., Hagan, S., Rath, O., Kolch, W., 2007. MAP kinase signalling pathwaysin cancer. Oncogene 26, 3279–3290.

Gao, B., Tsan, M.F., 2003. Endotoxin contamination in recombinant human heat shockprotein 70 (Hsp70) preparation is responsible for the induction of tumor necrosisfactor alpha release by murine macrophages. J. Biol. Chem. 278, 174–179.

Graner, M., Raymond, A., Akporiaye, E., Katsanis, E., 2000a. Tumor-derived multiplechaperone enrichment by free-solution isoelectric focusing yields potentantitumor vaccines. Cancer Immunol. Immunother. 49, 476–484.

Graner, M., Raymond, A., Romney, D., He, L., Whitesell, L., Katsanis, E., 2000b.Immunoprotective activities of multiple chaperone proteins isolated frommurine B-cell leukemia/lymphoma. Clin. Cancer Res. 6, 909–915.

Graner, M.W., Likhacheva, A., Davis, J., Raymond, A., Brandenberger, J., Romanoski,A., Thompson, S., Akporiaye, E., Katsanis, E., 2004. Cargo from tumor-expressedalbumin inhibits T-cell activation and responses. Cancer Res. 64, 8085–8092.

Graner, M.W., Zeng, Y., Feng, H., Katsanis, E., 2003. Tumor-derived chaperone-richcell lysates are effective therapeutic vaccines against a variety of cancers.Cancer Immunol. Immunother. 52, 226–234.

He, L., Feng, H., Raymond, A., Kreeger, M., Zeng, Y., Graner, M., Whitesell, L.,Katsanis, E., 2001. Dendritic-cell-peptide immunization provides immunopro-tection against bcr-abl-positive leukemia in mice. Cancer Immunol. Immun-other. 50, 31–40.

Heike, M., Weinmann, A., Bethke, K., Galle, P.R., 1999. Stress protein/peptidecomplexes derived from autologous tumor tissue as tumor vaccines. Biochem.Pharmacol. 58, 1381–1387.

Hennighausen, L., Robinson, G.W., 2008. Interpretation of cytokine signalingthrough the transcription factors STAT5A and STAT5B. Genes Dev. 22, 711–721.

Iwasaki, A., Medzhitov, R., 2004. Toll-like receptor control of the adaptive immuneresponses. Nat. Immunol. 5, 987–995.

Kawai, T., Akira, S., 2006. TLR signaling. Cell Death Differ.Klimp, A.H., de Vries, E.G., Scherphof, G.L., Daemen, T., 2002. A potential role of

macrophage activation in the treatment of cancer. Crit. Rev. Oncol. Hematol.44, 143–161.

Kryczek, I., Zou, L., Rodriguez, P., Zhu, G., Wei, S., Mottram, P., Brumlik, M., Cheng, P.,Curiel, T., Myers, L., Lackner, A., Alvarez, X., Ochoa, A., Chen, L., Zou, W., 2006.B7-H4 expression identifies a novel suppressive macrophage population inhuman ovarian carcinoma. J. Exp. Med. 203, 871–881.

Larmonier, N., Cantrell, J., Lacasse, C., Li, G., Janikashvili, N., Situ, E., Sepassi, M.,Andreansky, S., Katsanis, E., 2008a. Chaperone-rich tumor cell lysate-mediatedactivation of antigen-presenting cells resists regulatory T cell suppression.J. Leukoc. Biol. 83, 1049–1059.

Larmonier, N., Janikashvili, N., LaCasse, C.J., Larmonier, C.B., Cantrell, J., Situ, E.,Lundeen, T., Bonnotte, B., Katsanis, E., 2008b. Imatinib mesylate inhibits CD4+CD25+ regulatory T cell activity and enhances active immunotherapy againstBCR-ABL-tumors. J. Immunol. 181, 6955–6963.

Larmonier, N., Marron, M., Zeng, Y., Cantrell, J., Romanoski, A., Sepassi, M.,Thompson, S., Chen, X., Andreansky, S., Katsanis, E., 2007. Tumor-derivedCD4(+)CD25(+) regulatory T cell suppression of dendritic cell functioninvolves TGF-beta and IL-10. Cancer Immunol. Immunother. 56, 48–59.

Larmonier, N., Merino, D., Nicolas, A., Cathelin, D., Besson, A., Bateman, A., Solary,E., Martin, F., Katsanis, E., Bonnotte, B., 2006. Apoptotic, necrotic, or fusedtumor cells: an equivalent source of antigen for dendritic cell loading.Apoptosis 11, 1513–1524.

Li, Z., 1997. Priming of T cells by heat shock protein–peptide complexes as the basisof tumor vaccines. Semin. Immunol. 9, 315–322.

Li, Z., Menoret, A., Srivastava, P., 2002. Roles of heat-shock proteins in antigenpresentation and cross-presentation. Curr. Opin. Immunol. 14, 45–51.

McLaughlin, J., Chianese, E., Witte, O.N., 1987. In vitro transformation of immaturehematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphiachromosome. Proc. Natl. Acad. Sci. USA 84, 6558–6562.

Nathan, C., 1992. Nitric oxide as a secretory product of mammalian cells. Faseb J. 6,3051–3064.

Nicchitta, C.V., 2003. Re-evaluating the role of heat-shock protein–peptideinteractions in tumour immunity. Nat. Rev. Immunol. 3, 427–432.

ARTICLE IN PRESS

J. Cantrell et al. / Immunobiology 215 (2010) 535–544544

Pervin, S., Singh, R., Chaudhuri, G., 2001. Nitric oxide-induced cytostasis and cellcycle arrest of a human breast cancer cell line (MDA-MB-231): potential role ofcyclin D1. Proc. Natl. Acad. Sci. USA 98, 3583–3588.

Radsak, M.P., Hilf, N., Singh-Jasuja, H., Braedel, S., Brossart, P., Rammensee, H.G.,Schild, H., 2003. The heat shock protein Gp96 binds to human neutrophils andmonocytes and stimulates effector functions. Blood 101, 2810–2815.

Reed, R.C., Berwin, B., Baker, J.P., Nicchitta, C.V., 2003. GRP94/gp96 elicits ERK activationin murine macrophages. A role for endotoxin contamination in NF-kappa Bactivation and nitric oxide production. J. Biol. Chem. 278, 31853–31860.

Sanchez, P.J., McWilliams, J.A., Haluszczak, C., Yagita, H., Kedl, R.M., 2007.Combined TLR/CD40 stimulation mediates potent cellular immunityby regulating dendritic cell expression of CD70 in vivo. J. Immunol. 178,1564–1572.

Segal, B.H., Wang, X.Y., Dennis, C.G., Youn, R., Repasky, E.A., Manjili, M.H., Subjeck,J.R., 2006. Heat shock proteins as vaccine adjuvants in infections and cancer.Drug Discov. Today 11, 534–540.

Shuai, K., Liu, B., 2003. Regulation of JAK-STAT signalling in the immune system.Nat. Rev. Immunol. 3, 900–911.

Smith, D.F., Whitesell, L., Katsanis, E., 1998. Molecular chaperones: biology andprospects for pharmacological intervention. Pharmacol. Rev. 50, 493–514.

Srivastava, P., 2002a. Interaction of heat shock proteins with peptides and antigenpresenting cells: chaperoning of the innate and adaptive immune responses.Annu. Rev. Immunol. 20, 395–425.

Srivastava, P., 2002b. Roles of heat-shock proteins in innate and adaptiveimmunity. Nat. Rev. Immunol. 2, 185–194.

Srivastava, P.K., Amato, R.J., 2001. Heat shock proteins: the ‘Swiss Army Knife’vaccines against cancers and infectious agents. Vaccine 19, 2590–2597.

Srivastava, P.K., Menoret, A., Basu, S., Binder, R.J., McQuade, K.L., 1998. Heat shockproteins come of age: primitive functions acquire new roles in an adaptiveworld. Immunity 8, 657–665.

Srivastava, P.K., Udono, H., 1994. Heat shock protein–peptide complexes in cancerimmunotherapy. Curr. Opin. Immunol. 6, 728–732.

Tesselaar, K., Xiao, Y., Arens, R., van Schijndel, G.M., Schuurhuis, D.H., Mebius, R.E.,Borst, J., van Lier, R.A., 2003. Expression of the murine CD27 ligand CD70 invitro and in vivo. J. Immunol. 170, 33–40.

Wallin, R.P., Lundqvist, A., More, S.H., von Bonin, A., Kiessling, R., Ljunggren, H.G.,2002. Heat-shock proteins as activators of the innate immune system. TrendsImmunol. 23, 130–135.

Wells, A.D., Malkovsky, M., 2000. Heat shock proteins, tumor immunogenicity andantigen presentation: an integrated view. Immunol. Today 21, 129–132.

Xie, K., Dong, Z., Fidler, I.J., 1996. Activation of nitric oxide synthase gene forinhibition of cancer metastasis. J. Leukoc Biol. 59, 797–803.

Zeng, Y., Feng, H., Graner, M.W., Katsanis, E., 2003. Tumor-derived, chaperone-richcell lysate activates dendritic cells and elicits potent antitumor immunity.Blood 101, 4485–4491.

Zeng, Y., Graner, M.W., Katsanis, E., 2006. Chaperone-rich cell lysates, immuneactivation and tumor vaccination. Cancer Immunol. Immunother. 55, 329–338.

Zeng, Y., Graner, M.W., Thompson, S., Marron, M., Katsanis, E., 2005. Induction ofBCR-ABL-specific immunity following vaccination with chaperone-rich celllysates derived from BCR-ABL+ tumor cells. Blood 105, 2016–2022.