of June 18, 2018.This information is current as in a Beneficial Manner

Tolerance to CCL2 That Could Be Amplified Directs a Selective Loss of ImmunologicalTumor Site of Prostate Cancer Patients Predominant Expression of CCL2 at the

KarinJennifer Alami, Daniel Dumont, Avi Stein and Nathan Liat Izhak, Gizi Wildbaum, Weinberg Uri, Yuval Shaked,

http://www.jimmunol.org/content/184/2/1092doi: 10.4049/jimmunol.0902725December 2009;

2010; 184:1092-1101; Prepublished online 7J Immunol 

Referenceshttp://www.jimmunol.org/content/184/2/1092.full#ref-list-1

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Predominant Expression of CCL2 at the Tumor Siteof Prostate Cancer Patients Directs a Selective Lossof Immunological Tolerance to CCL2 That Could BeAmplified in a Beneficial Manner

Liat Izhak,* Gizi Wildbaum,* Weinberg Uri,* Yuval Shaked,† Jennifer Alami,‡ Daniel Dumont,‡

Avi Stein,x and Nathan Karin*

We have previously shown that, during inflammatory autoimmune diseases in humans, the immune system develops a neutralizing

auto-Ab–based response to a very limited number of inflammatory mediators, and that amplification of each response could be

beneficial for the host. Our working hypothesis has been that this selective breakdown of immunological tolerance is due to

a predominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. All three

conditions also take place in cancer diseases. In this study, we delineate this hypothesis for the first time in a human cancer disease

and then explore its clinical implications. We show that in primary tumor sections of prostate cancer subjects, CCL2 is pre-

dominantly expressed at the tumor site over other chemokines that have been associated with tumor development, including:

CXCL12, CXCL10, CXCL8, CCL3, and CCL5. Subsequently, the immune response selectivity mounts an Ab-based response to

CCL2. These Abs are neutralizing Abs. These findings hold diagnostic and therapeutic implications. The current diagnosis of

prostate cancer is based on prostate-specific Ag measurements that do not distinguish benign hypertrophy from malignancy. We

show in this study that development of anti-CCL2 Abs is selective to the malignant stage. From a clinically oriented perspective,

we show, in an experimental model of the disease, that DNA-based amplification of this response suppresses disease, which has

implications for a novel way of therapy in humans. The Journal of Immunology, 2010, 184: 1092–1101.

In previous studies that were initiated in experimental models

of different inflammatory autoimmune diseases and then

extended to humans, we have shown that in the course of

inflammatory autoimmune diseases, the immune system selectively

generates an auto-Ab response to a few inflammatory mediators,

mostly chemokines and cytokines, which are thought to participate

in promoting the inflammatory process (1–6). For example, we

showed that patients suffering from rheumatoid arthritis (RA) but

not osteoarthritis display a significant level of neutralizing auto-

Abs directed against TNF-a (3). These patients did not mount any

auto-Ab response either to several key inflammatory chemokines

or to regulatory mediators, such as IL-10 or TGF-b, or even the

chemokine CXCL12, which also functions as a regulatory medi-

ator that selects Ag-specific IL-10–producing CD4+ T cells (7).

Complementary experiments suggested that in experimentally in-

duced RA, these anti–TNF-a auto-Abs participate in the natural

regulation of disease and restrain—although they are incapable of

totally preventing—its development (3). Nevertheless, their selective

amplification by targeted DNA vaccines led to rapid recovery from

an ongoing disease (8). These studies also showed that selective

breakdown of tolerance to TNF-a is due to its preferential expres-

sion at a partially immune-restricted site undergoing a destructive

process (3, 8). Very recently, we have shown that type I diabetes

mellitus (T1DM) patients preferentially display auto-Ab production

to CCL3 and not to several other proinflammatory chemokines (9).

It is yet to be proven that this chemokine dominates the chemokine

expression at the autoimmune site. Nevertheless, a previous study

showing that selective neutralization of CCL3 suppresses T1DM in

NODmice has implications for the important role of this chemokine

in this disease (10).Similarly to organ-specific autoimmunity in various cancer

diseases, one of which is cancer of the prostate (CaP), key in-

flammatory chemokines are expressed at the primary tumor site,

which also undergoes a destructive process at a restricted site. This

motivated us to extend our research to CaP. Several recent studies

have shown that chemokines, in particular CXCL12 (SDF-1a),

CXCL8 (IL-8), CCL2 (MCP-1), CCL3, (MIP-1a), CCL5

(RANTES), and CXCL10 (IP-10), are produced at the tumor site

by the CaP cells that also express their receptors, and commonly

also by the supporting tissue (11–17). These chemokines are likely

to promote tumor development and angiogenesis (11–22).The current study shows that, of these chemokines, CCL2 is

dominantly expressed at the humanprimary tumor site and that these

patients selectively mount an auto-Ab response of neutralizing Abs

against CCL2. An immunocompetent model of the disease is then

used to explore the consequences of these findings.

*Department of Immunology and †Department of Pharmacology, the Ruth and BruceRappaport Faculty of Medicine, Rappaport Family Institute for Research in theMedical Sciences, Technion-Institute of Technology; xDepartment of Urology,Carmel Medical Center and the Ruth and Bruce Rappaport Faculty of Medicine,Haifa, Israel; and ‡Department of Medical Biophysics, University of Toronto, Tor-onto, Ontario, Canada

Received for publication August 18, 2009. Accepted for publication November 2,2009.

This study was supported by grants from the Israel Science Foundation.

Please address correspondence and reprint requests to Dr. Nathan Karin, Departmentof Immunology, Rappaport Family Institute for Research in the Medical Sciences,Technion-Institute of Technology, P.O.B. 9697, Haifa 31096, Israel. E-mail address:nkarin@tx.technion.ac.il

Abbreviations used in this paper: BPH, benign prostate hypertrophy; CaP, prostatecancer; CNBr, cyanogen bromide; PSA, prostate-specific Ag; RA, rheumatoid arthri-tis; T1DM, type 1 diabetes mellitus; VEGF, vascular endothelial growth factor.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902725

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Materials and MethodsSpecimens and Ab titers in human sera

The human experimental work was conducted together with the Departmentof Urology at Carmel Medical Center in Haifa, Israel. All sera and tissuesamples were obtained from patients according to Helsinki CommitteeApproval 0038-07-CMC, dated April 16, 2008. Clinical data of the patientsare summarized in Table I. Log2 Ab titer against each detected chemokinewas detected using an ELISA test on sequential sera dilution, as describedin detail below and in (3). All recombinant human chemokines werepurchased from R&D Systems (Minneapolis, MN).

Cell lines

All cell lines (PC-3, RAW, TRAMP-C1, and TRAMP-C3) were obtainedfrom the American Type Culture Collection (Rockville, MD).

In vitro chemotaxis assays

Chemotaxis assays of RAW cells (13 106) were performed in a TransWellsystem (5-mm pore size, Corning Costar Corporation, Cambridge, MA)(23). Chemotaxis assays of PC-3 and TRAMP-C1 cell lines (13 106) wereconducted using the CytoSelect TM cell migration assay (8-mm pore size)(Cell Biolabs, San Diego, CA) (23).

Evaluation of the Ab titer in sera samples

The titer of chemokine-specific Abs in the sera of CaP patients and miceinjected with TRAMP-C1 cells was determined using a direct ELISA and byusing two complementary measurement methods: comparing the ODmeasured in wells coated with the appropriate chemokine with those notcoated with this recombinant protein when sera were added in serialdilutions from 26–230 (log2X Abs titer), or direct measuring of OD aftera single dilution of 1:500 (9). All recombinant chemokines were purchasedfrom R&D Systems.

Cyanogen bromide purification of chemokine-specific Abs

Five milligrams recombinant chemokines (hCCL2, mCCL2) were bound toa cyanogen bromide (CNBr)-activated Sepharose column according to themanufacturer’s instructions (Pharmacia Biotech, Uppsala, Sweden, catalognumber 17-0820-01). Specific Abs from sera (IgG fraction) were loadedonto the column and eluted by an acidic elution buffer (glycine pH = 2.5).

Chemokine detection by ELISA

Detection of mCCL2 was performed by the ELISA development kit (mouseCCL2/JE ELISA kit, R&D Systems) and conducted according to themanufacturer’s instructions.

Animal models

C57BL/6 male mice were purchased from Harlen (Jerusalem, Israel) andmaintained in individual ventilated cages under pathogen-free conditionsin the animal facility of the Rapport Faculty of Medicine (Technion). Six-week-old mice were injected s.c. between the two flanks with 7 3 106

syngeneic TRAMP-C1 or TRAMP-C3. Mice were monitored daily forevidence of illness. Tumor diameters were measured using a caliper. Tu-mor volume was calculated using the formula p/6 3 a 3 b2, where a is thelongest dimension and b is the width. TRAMP mice (24) were bred andmaintained under pathogen-free conditions at the animal facility of theUniversity of Toronto, Toronto, Ontario, Canada. Results obtained fromthese mice were also verified (only weeks 8 and 10) using sera froma smaller colony of TRAMP mice obtained from the Animal Facility of theWeizmann Institute (Rehovot, Israel).

Immunohistochemistry and immunofluorescence

All immunohistochemistry and immunofluorescence staining and analyseswere conducted according to protocols we previously described (25, 26).For immunohistochemistry, the following Abs were used as primary Abs:polyclonal rabbit antivascular endothelial growth factor (VEGF) (sc-152,Santa Cruz Biotechnology, Santa Cruz, CA) and polyclonal rat anti-mouseF4/80 (MCA497B, Serotec, Raleigh, NC) for mouse macrophages. Forimmunofluorescence as primary Abs, we used mouse anti-hCCL2 (SantaCruz Biotechnology), mouse anti-hCXCL12 (R&D Systems), rabbit anti-hCXCL8 (PeproTech, Rocky Hill, NJ), rabbit anti-hCXCL10 (PeproTech),and rabbit anti-hCCL3 (PeproTech) all at 1:100 dilutions.

In vitro proliferation assay

In vitro proliferation assay of TRAMP-C1 cells was conducted as wedescribed in detail in (23).

DNA vaccination

The cDNA encoding mouse CCL2 and b-actin were obtained from spleno-cytes of naive mice and subjected to RT-PCR amplification of the CCL2 andthe b-actin–encoding genes using the oligonucleotide primers (forward 59-ATGCTTGGCTCAGCAC-39, reverse 59-TCAATTTTTCATTTTGAGTGT-39) and b-actin (forward 59-TTCTTTGCAGCTCCTTCGTTGCCG-39, re-verse 59-TGGATGGCTACGTACATGGCTGGG-39). After sequencing,verification PCRproductswere transferred into a pcDNA3vector (Invitrogen,San Diego, CA). Large-scale preparation of plasmid DNA was conductedusingMegaPrep (Qiagen, Chatsworth, CA). Cardiotoxin (Sigma-Aldrich, St.Louis,MO)was injected into the anteriormuscle of the tibias of 5- to 6-wk-oldC57BL/6 mice (10 mM per leg). One week after injection, the mice wereinjected with 100 mg DNA in PBS.

RNA extraction and real-time PCR

Prostate tissue samples were stored in liquid nitrogen until RNA extraction.RNA was extracted using TriReagent (Sigma-Aldrich), according to themanufacturer’s instructions, and was reverse transcribedwithM-MLV reversetranscriptase (Promega, Madison, WI) using random primers (AmershamBiosciences, Piscataway, NJ). Quantitative PCRwas preformedwithAbsoluteBlue SYBR-Green ROX Mix (Thermo Scientific, ABgene, Hamburg,Germany), according to the manufacturer’s instructions, with the Rotor-Gen-eTM 6000 system (Corbett Research, Sydney, Australia) and its software,version 1.7. The amounts of transcripts were normalized to that of b-actin.Melting curveswere determined to ensure the amplification of a single product.The primers used were: hCCL2, F9-CAGCCAGATGCAATCAATGCC9,R9-TGGAATCCTGAACCCACTTCT9; hCCL3, F9-AGTTCTCTGCATCA-CTTGCTG9, R9-CGGCTTCGCTTGGTTAGGAA9; hCCL5, F9-ATCCTCA-TTGCTACTGCCCTC9, R9-GCCACTGGTGTAGAAATACTCC9; hCXCL8,F9-ACTGAGAGTGATTGAGAGTGGAC9, R9-AACCCTCTGCACCCAGT-TTTC9; hCXCL10, F9-GTGGCATTCAAGGAGTACCTC9, R9-GCCTTCG-ATTCTGGATTCAGACA9; hCXCL12, F9-ATGCCCATGCCGATTCTTCG9,R9-GCCGGGCTACAATCTGAAGG9; b-actin, F9-CATGTACGTTGCTATC-CAGGC9, R9-CTCCTTAATGTCACGCACGAT9.

Formouseprimarytumor,weusedthesamebasicprotocol,yet theamountsof transcriptswere normalized toGAPDH.The following primerswere used:mouseCCL2, F9-TTAAAAACCTGGATCGGAACCAA9, R9-GCATTAGC-TTCAGATTTACGGGT9; mouse GAPDH, F9-CATGTTCCAGTATGACT-CCACTC9, R9-GGCCTCACCCCATTTGATGT9; mouse CCL5, F9-GCTG-CTTTGCCTACCTCTCC9, R9-TCGAGTGACAAACACGACTGC9; mouseCXCL12, F9-GTCAGCCTGAGCTACCGATG9, R-9TTCTTCAGCCGTG-CAACAATC9.

Macrophage depletion

Clodronate (dichloromethylene diphosphonate)-induced macrophage de-pletion was conducted according to Jordan et al. (27). The depletion ofmacrophages was verified by flow cytometry analysis on spleen cells (F4/80 Ab, BD Biosciences, San Jose, CA). The depletion was consideredsuccessful only if $98% of these cells were depleted.

Western blot analysis

Western blot analysis was conducted as described in (11).

Statistical analysis

The significance of differenceswas examinedusing theStudent t test;p,0.05was considered statistically significant. Significance of Ab titer was de-termined using receiving operating characteristic curve analysis that is basedon the binomial distribution to obtain upper and lower bounds for 95% con-fidence intervals surrounding estimates of test sensitivity and specificity (28).

ResultsCaP patients display a significant auto-Ab titer to CCL2

Our study included 23 CaP patients, 21 individuals with benignprostate hypertrophy (BPH), and 11 control subjects (Table I) whowere tested for the expression of different chemokines (immu-nofluorescence analyses of prostate gland sections and real-timePCR) and the development of auto-Abs to these chemokines asdetermined by ELISA on sera samples. In all subjects, sera were

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taken before entering any therapeutic protocols (i.e., radical

prostatectomy, hormone therapy, or radiation).We first subjected human malignant primary tissues from five

different patients with Gleason scores varying from 4–7 to immu-

nofluorescence analyses for the expression of CCL2, CCL3, CXCL8,

CXCL10, and CXCL12, and compared themwith reciprocal samples

from five BPH subjects (five sections from each subject). Fig. 1A

displays representative sections subjected to comparative staining of

these chemokines. Apparently, CCL2 dominates the chemokine ex-

pression within the malignant tissue (Fig. 1Aa compared with 1Ac,

1Ae, 1Ag, and 1Ai). Notably, sections from BPH subjects displayed

relatively lower expression ofCCL2 comparedwithmalignant tissues

(Fig. 1Ab comparedwith 1Aa). Computerized analyses of all sections

(2035 fromeitherCaPorBPH subjects) showed significantly higher

fluorescence intensity (as determined in pixels per field) of CCL2

staining in sections from CaP subjects compared with each of the

other chemokines (Fig. 1B) (4.156 0.9 versus 0.096 0.005, 0.2160.06, 1.556 0.4, 2.176 0.6, p, 0.01, for the comparison of CCL2

staining to CCL3, CXCL10, CXCL8, and CXCL12 in CaP subjects,

respectively) and also in comparisonwithCCL2 staining inBPH (Fig.

1B) (4.16 0.9 versus 0.536 0.07, p, 0.01). Fig. 1B also shows that

the fluorescence intensity of CCL2 in CaP is ∼10-fold higher than inBPH. Within the BPH group, however, the expression of this che-

mokinewas significantly higher than CCL3, CXCL10, and CXCL12

(Fig. 1B, p, 0.01).A similar pattern of results was obtained when determining the

transcription of each chemokine in tissue samples of CaP subjects

(calibrated to b-actin) (Fig. 1C) (8.36 1.1 versus 1.26 0.4, 3.960.5, 0.56 0.05, 46 0.9, 4.76 0.8; p, 0.01, for the comparison of

CCL2 transcription to the transcription of CCL3, CCL5, CXCL10,

CXCL8, andCXCL12 inCaP subjects, respectively) and also for the

FIGURE 1. Selective breakdown of tolerance to

CCL2 occurs during the malignant stage of CaP. A

and B, Immunofluorescence analysis was applied

on sections from the prostate of subjects with ma-

lignancy (A, a, c, e, g, and i) or BPH (A, b, d, f, h,

and j). Sequential sections were stained with either

anti-CCL2 (a, b), anti-CCL3 (c, d), anti-CXCL8 (e,

f), and anti-CXCL10 (g, h) or anti-CXCL12 (i, j)

(original magnification 320). A representative set

of sections of CaP subjects with a Gleason score of

6 and a BPH subject are shown. Full analysis of all

sections from the different subjects was also de-

termined and is shown in (B). Five random micro-

scopic fields from three different sections were

examined for fluorescence intensity (pixels per

field). The intensity was measured as mean of the

relative number of bright pixels per field as mea-

sured in five different fields per section (B). C,

Real-time PCR analyses for the transcription of

CCL2, CCL3, CCL5, CXCL10, CXCL8, and

CXCL12 in prostate tissue from five malignant

subjects and five BPH subjects. All samples were

calibrated to the transcription of b-actin. Tissue

samples were selected after histopathological veri-

fication of CaP and BPH. Results are shown as

relative expression 6 SE. D, Sera from 23 CaP

patients, 21 BPH, and 11 control subjects were

tested for the presence of auto-Ab to CCL2, CCL3,

CCL5, CXCL8, CXCL10, and CXCL12 by direct

ELISA (a), or OD (450 nm) at a single dilution of

1:500 (b). Results are shown either as mean log2 Ab

titer 6 SE (a), or mean OD 6 SE (b). E, Inhibition

of CCL2-induced migration of PC-3 cells by CNBr-

chemokine-purified auto-Ab to CCL2. The chemo-

taxis index was determined in the absence or

presence of 20 ng/ml CCL2 and 50 mg/ml purified

Abs or anti-CCL2 mAb. Results are shown as mean

triplicates 6 SE.

Table I. Patient demographics and characteristics

Number of CaP patients enrolled 23Age, y [median (range)] 74 (64–81)PSA, ng/ml [median (range)] 6.8 (3.7–3400)Gleason score#6 37 18

$8 2Number of BPH patients enrolled 21Age, y [median (range)] 71 (64–79)PSA, ng/ml [median (range)] 3.4 (2.3–8)Number of healthy controls enrolled 10Age, y [median (range)] 70 (62–75)

The basic clinical information regarding CaP patients who participated in thestudy is summarized. It complements the data presented in Fig. 1. In all CaP patientsincluded in Table I and Fig. 1, CCL2 titers and PSA levels were obtained beforetreatment. All patients except for one (metastatic patient with a PSA level of 3400)are patients with clinically localized disease (stages T1a, T1b, and T2).

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comparison of CCl2 transcription in CaP subjects compared withBPH subjects (Fig. 1C) (8.3 6 1.1 versus 2.1 6 0.6, p, 0.01).Then, sera from all 23 CaP patients, 21 individuals with BPH,

and 11 control subjects were tested for the presence of auto-Abs toCCL2 (MCP-1), CCL3 (MIP-1a), CCL5 (RANTES), CXCL8(IL-8), CXCL10 (IP-10), and CXCL12 (SDF-1a). Of these che-mokines, CaP patients mounted a highly significant Ab titer ex-clusively to CCL2 (Fig. 1Da, log2 Ab titer of 11.85 6 0.8). Thebaseline titer of anti-CCL2 Abs (log2) in healthy individuals andin BPH patients was ∼6 (p , 0.001, compared with anti-CCL2)and did not differ from the one observed in response to any of theother chemokines. A complementary examination at a single seradilution of 1:500 revealed an exclusive 3.5-fold increase in OD(450 nM) of anti-CCL2 Ab production only in sera of CaP sub-jects (Fig. 1Db, p , 0.0001 compared with all other groups).Comparative analysis of CCL2-specific Ab titer developed in

CaP compared with BPH and age-matched control subjects wasalso conducted. Results are shown as log2 Ab titer. The cutoff (log2Ab titer .9) was determined using ROC curve analysis as de-scribed in Materials and Methods. Thus, 82% (19/23) of CaPpatients and 4.7% (1/21) of BPH patients displayed a significantresponse to CCL2. Taken together, these results show that selec-tive loss of tolerance to a chemokine may occur if this chemokineis predominantly expressed at a tumor site. We have previouslyshown in experimental models of autoimmunity, which were ex-tended to humans, that during inflammatory autoimmunity, but notin response to bacterial adjuvants, the immune system selectivelybreaks down the tolerance to key inflammatory mediators that arepredominantly expressed at the autoimmune site, such as TNF-ain RA patients (3), which is predominantly expressed at the in-flamed joint and is a successful target for therapy (29). Takentogether, these data and our current observations suggest that,within a segregated tissue undergoing a destructive process, theimmune system generates an Ab-based response to inflammatorymediators that are predominantly expressed within the tissue.CaP cells produce CCL2 and express its CCR2 receptor (30).

Targeted neutralization of CCL2 is thought to be beneficial inblocking tumor development and its spread (12, 13). As CaP pa-tients do display an apparent anti-CCL2 Ab titer, it is an open-ended question whether these Abs participate in the natural reg-ulation of the disease. The minimal requirement for such a possi-ble beneficial involvement is the ability to neutralize CCL2. Todetermine this possibility, we purified sera of the previously de-scribed 23 CaP patients on a CNBr-CCL2 chemokine purificationcolumn and tested the purified Abs for their ability to inhibitCCL2-induced migration of the human CaP cell line PC-3. Fig. 1Eshows that these Abs neutralized and significantly inhibited theCCL2-induced migration of this line (p , 0.001). These ob-servations motivated us to further explore the role of anti-auto–Absagainst CCL2 in the regulation of CaP, and their amplification asa potential therapeutic intervention.

Elicitation of auto-Ab production of CCL2 inimmunocompetent mice is dependent on cancer celltumorigenecity

TRAMP, developed by Dr. N. Greenberg, is a spontaneously de-veloped disease in transgenic mice (C57BL/6 background) (24).Three CaP cell lines, termed C1, C2, and C3, were generated fromthese mice by Dr. Greenberg (24). Of these lines, C1 very effec-tively forms tumors in immunocompetent mice, whereas C3 lacksthis ability (31). Before administering C1 and C3 lines, we verifiedthat both produce CCL2 in cultured media (ELISA) and expressits CCR2 receptor (FACS analysis, rabbit anti-mouse CCR2 [E68],Novus Biologicals, Littleton, CO). Both lines were administered

to immunocompetent C57BL/6 mice. In accordance with Foster

et al. (31), only C1 successfully formed an established primary

tumor with a time-dependent increase in tumor volume (Fig. 2A),

with apparent tumor angiogenesis, as verified by histological

evaluation (not shown). Both lines produced (in vitro) comparable

levels of CCL2 (not shown). Yet, real-time PCR analysis con-

ducted on primary tumor sections isolated on day 25 from mice

administered with the C1 line (Fig. 1A) showed a marked increase

in the transcription of CCL2 (p , 0.0001) compared with those

injected with the C3 line (Fig. 2B). Moreover, mice developing

FIGURE 2. Development of the primary tumor in immunocompetent

mice is associated with a selective breakdown of tolerance to CCL2. A,

Two groups of C57BL/6 mice were injected with 7 3 106 TRAMP-C1

cells or TRAMP-C3 cells and monitored weekly for the development of

primary tumor. Results are shown as tumor volume 6 SE. The data pre-

sented here represent one of three independent experiments with similar

data. B, Relative transcription of various chemokines (normalized to

GAPDH) as determined by real-time PCR on samples of primary tumors

(A, day 25) developed after C1 or C3 cell administration, as determined by

real-time PCR. Results of six samples from three different mice per group

are shown as mean 6 SE. C, Sera from naive mice and mice injected with

TRAMP-C1 cells or TRAMP-C3 cells were tested for the presence of auto-

Ab to various chemokines. Results are shown as mean log2 Ab titer 6 SE

of six mice per group. The vast majority of mice injected with C1 cells

developed auto-Ab to CCL2. D, The kinetics of CCL2-specific auto-Ab

titer developed in groups of mice injected with 7 3 106 C1 or C3 cells/

mouse s.c., as described in (A). Results are shown as mean log2 Ab titer 6SE of six mice per group. E, Inhibition of CCL2-induced chemotaxis of

RAW cells by purified Abs from mice administered with C1 cells (50 mg/

ml CNBr-CCL2–purified Abs), compared with control IgG purified from

healthy age-matched C57BL/6 donors, and commercially available anti-

CCL2 neutralizing Ab. None of the Abs could inhibit CCL5-induced mi-

gration of these cells (not shown). Results are shown as the mean che-

motaxis index of triplicates 6 SE.

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C1-induced tumor elevation in the transcription of other chemo-

kines, including CCL5 and CXCL12, displayed only a minor in-

crease compared with CCL2 (p , 0.001). Subsequently, only C1

that successfully formed an established primary tumor also initi-

ated the development of specific auto-Abs to CCL2, but not

CCL3, CCL5, CXCL10, or CXCL12, as presented by Fig. 2C (25

d after tumor implementation, log2 Ab titer of 116 0.5 versus 660.5 and 7 6 1.66 in control groups injected with C3 or PBS, p ,0.001). This CCL2-specific high Ab titer continued to persist

along tumor development and progression (Fig. 2D) in the vast

majority of detected mice (16/18 in three independent experi-

ments). We then purified these Abs (affinity purification), and

determined their ability to inhibit the migratory properties of

RAW cells in response to CCL2. Fig. 2E shows that these CCL2-

specific Abs could markedly (p , 0.0001) inhibit the migration of

these cells in a trans-well system in a similar manner to our

control anti-CCL2 mAb (R&D Systems). Both Abs could not

inhibit CCL5 induced migration of RAW cells (not shown).

Taken together, these results show that in humans and in a murinesetting, the development of a tumor may lead to selective break-down of tolerance that is dependent on the tumorigenicity of thetransformed malignant cells.

CCL2-encoding DNA vaccine amplifies the production ofspecific auto-Abs against CCL2 and suppresses tumordevelopment

Because of the relevance to humans, we determined whether tar-geted DNA plasmid encoding CCL2 could enhance Ab productionto its gene product in mice displaying anti-CCL2 Ab titer and, if so,whether this amplification affects the dynamics of tumor de-

velopment. Fig. 3 summarizes the results of one of three differentexperiments with similar results, in which groups of six mice wereinjected with the 7 3 106 C1 cell line and 25 d later, when theprimary tumor was established (verified also histologically inrepresentative mice). These mice were injected every 5 d with

CCL2-encoding DNA plasmid, b-actin–encoding DNA plasmid,

FIGURE 3. CCL2-encoding plasmid DNA am-

plifies auto-Ab production to CCL2 and suppresses

the development of the primary tumor. A, The ki-

netics of CCL2-specific auto-Ab titer developed in

groups of mice injected with 7 3 106 C1 cells/

mouse s.c., which were or were not injected 25

d later, to repeated injections (100 mg DNA in PBS,

every 5 d) of pcDNA-CCL2, pcDNA-b–actin or

PBS, compared with naive mice. Results are shown

as mean log2 Ab titer 6 SE of six mice per group.

B, Mice of the above groups were monitored for the

growth of the primary tumor every 5 d by an ob-

server blind to the experimental protocol. Results

of one of three experiments, with similar data, are

shown as mean six mice6 SE. Tumor development

in pcDNA-CCL2–vaccinated mice was signifi-

cantly reduced (p , 0.001). C, On day 70, the mice

were euthanized and subjected to immunofluores-

cence and immunohistochemical analyses using

anti-CD31 (a–c, frozen sections), anti-F4/80 (d–f),

and anti-VEGF (g–i) (original magnification 340).

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or PBS. Fig. 3A shows that as early as 10 d after the first plasmidDNA injection, those mice injected with CCL2-encoding DNAdeveloped an accelerated Ab titer against CCL2 (Fig. 3A, day 35,log2 Ab titer of 17.5 6 1.6, 9.4 6 1.1 and 10.8 6 0.7, re-spectively), but not against each of the other chemokines shown inFig. 2C (not shown). This titer continued to persist during theprogression of disease (Fig. 3A). An observer blind to the exper-imental protocol monitored the progression of the primary tumor.Fig. 3B summarizes the results of one of three experiments withsimilar data, showing that only the administration of CCL2-encodingDNA plasmid significantly slowed tumor development (e.g., on day45, a tumor volume of 706 10mm3 versus 4106 36mm3 and 410630 mm3; p , 0.001). We also determined the level of CCL2 in sera(R&D Systems, ELISA kit) of mice subjected to CCL2-encodingDNAvaccine, comparedwith controlmice developing C1 tumor (day25). Our results show a 2–3-fold increase in sera levels of CCL2 inDNAvaccinatedmice. These results are comparable to thosewe haverecently reported, in which CCL2 was neutralized (in vivo) by a sol-uble receptor (23).On day 70, all mice were euthanized and subjected to immu-

nofluorescence and immunohistochemical analyses. As shown inFig. 3C, repeated injection of DNA vaccine to CCL2 led toa marked reduction in CD31 staining (for vessels) (Fig. 3Caversus 3Cb and 3Cc) as well as F4/80+ macrophages (Fig. 3Cdversus 3Ce and 3Cf), in accordance with decreased VEGF ex-pression at the tumor site (Fig. 3Cg versus 3Ch and 3Ci).

CCL2-specific Abs isolated from protected donors transfer thebeneficial effect of plasmid DNA therapy

The association between the acceleration of CCL2-specific Ab titerafter the administration of CCL2-encodingDNAplasmid and tumorregression may suggest that these Abs suppress tumor developmentby neutralizing CCL2. In attempting to evaluate this hypothesis, wepurified these Abs from protected donors (Fig. 3, day 70) and, afterCNBr-Ag purification, we determined their in vitro neutralizingproperties and then their competence to suppress the development ofC1-induced primary tumor in immunocompetentmice (Fig. 4).CCL2is a chemoattractant for the tumor cells and for tumor-associatedmacrophages, which are likely to facilitate tumor development. Wefirst detected the ability of our Abs to affect the CCL2-induced mi-gration of a commercially available macrophage cell line, RAW. Asshown in Fig. 4A, anti-CCL2 Abs purified from protected donors, butnot from those mice injected with b-actin–encoding DNA plasmid(only IgG purification) could markedly (p, 0.001) suppress CCL2-induced migration of RAW cells similarly to commerciality availableAbs against CCL2. Subsequently, these Abs, but not those purifiedfrom mice injected with the b-actin–encoding plasmid, inhibited theCCL2-inducedmigration of the tumor C1 cells (Fig. 4B, p, 0.0001).Before their in vivo administration, we examined the ability of theseAbs to inhibit the in vitro proliferation of C1 cells (Fig. 4C). Thus,purified anti-CCL2 Ab from donors with reduced tumor progressioninhibitedmacrophage and tumor cellmigration and also tumorgrowth(proliferation). What is the mechanistic basis by which CCL2 affectsthese functions of C1 cells? Previous studies conducted on the humanPC3 line showed that CCL2 activates the PI3K/AKT pathway re-sulting in the phosphorylation of p70-S6 kinase (a downstream targetof AKT) and induced actin rearrangement, resulting in a dynamicmorphologic change indicative of microspike formation (11). Veryrecently, Roca et al. (32) showed that CCL2 maintains the survival ofthese cells via PI3K/AKT-dependent pathway. The current studyshows thatCCL2-induced signalingonC1cells also includes the samepathway (Fig. 4D), which is CCL2-induced AKT phosphorylationthat could be inhibited by Wortmannin: a fungal metabolite that

specifically inhibits PI3K, MAPK, and myosin light-chain kinase(33), and P70 S6k phosphorylation that could be inhibited byLY294002: a specific inhibitor of PI3K, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (34).Finally, we determined the ability of these Abs to suppress C1

cell-induced tumor development. Fig. 4E shows that repeatedadministration (every 5 d, 200 mg/mouse, i.v.), beginning on day20 when tumor size was 21.6 6 5.5 mm3, slowed tumor de-velopment (e.g., day 60 tumor volume of 170 6 26 mm3 versus600 6 52 mm3 and 680 6 71 mm3, p , 0.001). These resultsstrongly suggest that targeted DNA plasmids encoding CCL2amplify beneficial anti-CCL2 auto-Ab production during tumordevelopment and, by so doing, suppress its progression.The association between targeted neutralization ofCCL2 (Figs. 3,

4), inhibition of tumor associated macrophages recruitment at thetumor site (Fig. 3C), and inhibition of tumor development maysuggest that targeted neutralization of CCL2 affect tumor de-velopment, in part, by inhibiting macrophage recruitment at thetumor site. To further investigate this hypothesis, we have conductedan additional set of experiments in which three groups of mice wereadministered with 73 106 C1 cells and, 7 d later, subjected to eitherneutralization of CCL2 by a plasmid DNA vaccine encoding thischemokine or to a depletion of macrophages by repeated injectionsof clodronate liposomes in a 4-d interval (Roche DiagnosticsMannheim, Germany). Effectiveness of macrophage depletion wasdetermined using additional mice that were treated with clodronateliposomes and sacrificed on days 8 and 12. Macrophage depletionwas considered successful only if$98%were depleted (using a flowcytometry analysis). Fig. 5A shows that although mice treated withPBS (C1 group) and those administrated with an empty liposomecontinued to develop a progressive primary tumor (day 55, meantumor volume of 4906 50 mm3 and 5306 55 mm3), those treatedwith either CCL2-encoding DNA plasmid or clodronate liposomesdisplayed a markedly reduced tumor development (170 6 20 mm3

and 2306 25mm3, respectively, p, 0.001 in comparisonwith eachcontrol group). Complementary immunostaining (F4/80) of primarytumor sections of each group (Fig. 5B, day 55) showed a markeddecrease in the accumulation of tumor-associated macrophages inmice subjected to either CCL2-encoding plasmid DNA, or macro-phage depletion (Fig. 5Bc and d comparedwith 5Ba and 5Bb). Takentogether, these results suggest that therapy with plasmid DNA en-coding CCL2 leads to increased CCL2-specific Ab production,which inhibits the recruitment of tumor-associated macrophages atthe tumor site, and that these macrophages are essential to supporttumor development.

DiscussionWe have previously shown in experiments that were initiated inexperimental models and extended to humans that, during in-flammatory autoimmune diseases, the immune system developsa neutralizing auto-Ab–based response to a very limited number ofinflammatory mediators (3). It is tempting to assume that thisresponse is initiated to restrain the harmful consequences of thesediseases. Nevertheless, it is clear that decisions taken by the im-mune system are based on simplistic rules of interactions, ratherthan rational thinking. Our working hypothesis has been that thisselective breakdown of immunological tolerance is due to a pre-dominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. If so, such con-ditions also characterize cancer diseases, in which inflammatorymediators are highly expressed at the primary tumor site, whichundergoes an immune-restricted destructive process. At the tumorsite CCL2 is produced by the cancer cells, invading macrophages,and by the endothelium, all of which express its CCR2 receptor

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(11, 30). We show in this study that targeted neutralization of

CCL2 inhibited macrophage (F4/80+) cell accumulation at the

tumor site in association with decreased angiogenesis (determined

by CD31 staining) and decreased VEGF could be recorded (Fig.

3C), which may explain, in part, the inhibition in tumor growth.

Nevertheless, we do not have direct proof for the working hy-

pothesis that the blockade of CCL2 directly inhibits macrophage

accumulation at the tumor site. We also do not know whether

FIGURE 4. CCL2-specific auto-Abs produced in DNA-vaccinated mice transferred the beneficial effect of the vaccine. A, Inhibition of CCL2-induced

chemotaxis of RAW cells by purified Abs compared with commercially available anti-CCL2 neutralizing Ab. The ability of CCL2-specific auto-Abs (50

mg/ml CNBr-CCL2–purified Abs) from DNA-vaccinated mice to inhibit CCL2-induced migration compared with those purified from mice injected with

b-actin–encoding DNA or commercially available anti-CCL2 neutralizing Ab compared with CCL5-induced migration. Results are shown as the mean

chemotaxis index of triplicates 6 SE. B, CNBr-CCL2–purified Abs inhibits CCL2-induced migration of TRAMP-C1 cells. The ability of CCL2-specific

auto-Abs (50 mg/ml CNBr-CCL2–purified Abs) from DNA-vaccinated mice to inhibit CCL2-induced migration was examined in a CytoSelect cell mi-

gration assay (8-mm pore size) compared with those purified from mice injected with b-actin–encoding DNA or commercially available anti-CCL2

neutralizing Ab. Results are shown as mean triplicates 6 SE. C, The ability of CCL2-specific auto-Abs developed in mice injected with CCL2-encoding

DNA plasmid to inhibit the proliferative response of C1 was compared with cultures supplemented with PBS (medium), with b-actin–encoding DNA

plasmid, or with the addition of commercially available anti-CCL2 Ab. Results are shown as mean [3H]thymidine uptake of triplicates6 SE. D, The ability

of CCL2 to induce the phosphorylation of AKT and P70-S6k in C1 cells, and of Wortmannin and LY294002 to inhibit these phosphorylations. E, Three

groups of six C57BL/6 mice were injected with 73 106 C1 cells/mouse s.c. and treated every 5 d, starting day 20, with PBS, 200 mg/ml purified anti-CCL2

Ab, or 200 mg/ml purified anti-b–actin Ab. Mice were monitored every 5 d for the development of the primary tumor by an observer blind to the ex-

perimental protocol. Results are shown as mean 6 SE of six mice per group.

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reduced VEGF and possibly other growth factors at the tumor sitesof these mice is due to a direct effect of macrophages that arelikely to produce them at the tumor site, or due to their interactionwith other cells there. Fig. 5 shows that direct depletion of mac-rophages also inhibited tumor growth, partially supporting thehypothesis that these cells support tumor growth either directly orbecause of their interaction with other cells at the tumor site,.We show that of the various chemokines associated with CaP,

CCL2 is predominantly expressed at the primary tumor site of thesepatients, and that they develop a selective neutralizing auto-Ab titerin response to CCL2. We then used an immunocompetent model ofthe disease to show that targeted amplification of this response ledto tumor regression, thus demonstrating a practical way of com-bating the disease.Lu et al. (12) showed in 2006 that CCL2 expression correlates

with pathology in human CaP. These results are in accordancewith our current study (Fig. 1D, 1E). Using a xenograft model ofCaP in SCID mice, the same group showed in 2007 that Ab-basedblockade of CCL2 induces tumor regression in vivo (13). Sub-sequently, very recently, we published similar observations usingan Ig-CCR2–based fusion protein that preferentially binds andneutralizes CCL2 (23). These results, together with those of thecurrent manuscript (Fig. 4), suggest that CCL2 is likely to be animportant target for therapy for CaP. Selective breakdown of tol-erance to this chemokine in humans (Fig. 1) may suggest an ap-plicative way of therapy for CaP patients. The current study showsthat patients who have CaP display an apparent auto-Ab titer ofneutralizing Abs against CCL2 that are likely to participate in theregulation of disease in a beneficial manner. These Abs could beused either for diagnosis of the malignant stage of disease, or as

a target for beneficial amplification. The mechanistic basis of howthe immune system selectively breaks down tolerance to chemo-kines that are preferentially expressed at the tumor site is far frombeing understood. Our working hypothesis is that under theseconditions at first auto-reactive CD4+ T cells are activated in re-sponse to this chemokine. Then, these cells further activate che-mokine-specific B cells. This hypothesis has still to be proved.Fig. 1 also shows that the transcription of several chemokines

including CCL2, CCL5, CXCL12, and CXCL8 is selectively el-evated in CaP compared with BPH. At the tumor site, thesechemokines are likely to be produced by various types of cells, inparticular the cancer cells, tumor-associated macrophages, andother bone marrow-derived cells that preferentially accumulate atthe tumor site during malignancy (35). This may explain the ac-cumulation in chemokine transcription and expression duringmalignancy and not BPH.Production of auto-Abs to two proinflammatory chemokines

CCL2 and CXCL8 after i.v. administration of LPS was previouslyreported (36). LPS administration leads to a rapid elevation inchemokine level in the sera, which is likely to assist this break-down of tolerance.Over the last 10 y, we have shown in experimental models of

several inflammatory autoimmune diseases that targeted DNAplasmids encoding inflammatory cytokines/chemokines (1, 4–6, 8,37–39). Throughout these studies we noticed that these vaccines,when injected after induction of disease, act very rapidly, becausethey amplify a pre-existing response initiated by the autoimmuneprocess (1–3, 8, 39). Nevertheless, the mechanistic basis of howthe immune system generates a selective breakdown of toleranceunder an autoimmune condition, but not after adjuvant-inducedlocal inflammatory response (3), or how it restricts the response toa very limited number of mediators, remains elusive. One mustunderstand that this system is incapable of making its own rationaldecisions regarding what could be beneficial for the regulation ofan autoimmune process or malignancy.We have previously shown that in RA, the immune system of

these patients selectively mounts an auto-Ab response to TNF-a (3)that is predominantly expressed at the autoimmune site (40). Veryrecently, we showed that during T1DM, diabetic subjects displaya selective auto-Ab response to CCL3 (9). We think that in hu-mans this chemokine is predominantly expressed at the autoim-mune site, although the inaccessibility of human pancreas samplesfrom these patients precludes indepth examination of this as-sumption. A common denominator of tumor development andautoimmunity is that both include a “pathogen-free” destructiveprocess, in which inflammatory mediators, including chemokinesare highly expressed at an immune-restricted site. Similar con-ditions occur in solid cancers. This motivated us to expand ourstudy to cancer diseases, where the accessibility of primary tumorsections from human subjects is more accessible. The currentstudy, using human cancer primary tissue samples, together withour early publication using RA samples, (3) imply that, regardlessthe nature of disease, the predominant expression of an in-flammatory mediator at an immune-restricted site, under de-structive conditions, would provide appropriate conditions forselective Ab-based loss of immunological tolerance.Even though CaP patients do display an apparent anti-CCL2 Ab

titer (Fig. 1A) that should protect them, they develop a progressivedisease. Why so? It is likely that as neutralizing Abs, they inducea beneficial effect, but their low titer is perhaps sufficient to re-strain but not fully suppress its development and progression.The role of CCL2 in cancer, particularly CaP and other androgen-

dependent cancer diseases, could be viewed from two comple-mentary perspectives: its direct effect on recruitment and growth of

FIGURE 5. Depletion of macrophages suppresses the development of the

primary tumor. A, Three groups of mice were administered with 73 106 C1

cells, and 7 d later subjected to either neutralization of CCL2 by a plasmid

DNA vaccine encoding this chemokine (closed circles), or to depletion of

macrophages by repeated injections of clodronate liposomes, in a 4-d in-

terval (closed square). Controlmicewere treatedwith PBS (open circules) or

an empty liposome (closed triangles). Micewere monitored every 5 d for the

development of the primary tumor by an observer blind to the experimental

protocol. Results are shown as mean 6 SE of six mice per group. B, Com-

parative immunostaining of macrophages (F4/80) at the primary tumor (day

55) of the primary tumor of mice injected with either: PBS (a), empty lip-

osomes (b), plasmid-encoding CCL2 (c), or clodronate liposomes (d). Each

section represents 18 different sections of 6 mice per group.

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malignant cells (12, 13), and its role in recruiting tumor-associatedmacrophages to assist tumor invasion (41–45). Focusing on the roleof the CCL2–CCR2 interaction in macrophage recruitment at thetumor site, we show in this study that, indeed, targeted neutrali-zation of CCL2 inhibits the migration of tumor-associated macro-phages to the tumor site, and at the same time, leads to markedlydecreased expression of the major angiogenic factor VEGF, whichdirects tumor implementation (46).One possible ramification of our study is its potential extrapo-

lation in humans. This strategy holds advantages and drawbackscompared with Ab or soluble receptor-based therapies. The majoradvantages are that it interferes, by amplification, a natural processof targeted neutralization. Such amplification could lead to auto-development of improved therapy because of affinity maturation ofthe chemokine-specific Abs, although this hypothesis has yet to beproven. The two major drawbacks of DNA-based vaccines, aimedat amplifying beneficial immunity are as follows: 1) Once am-plified, such a response is regulated, independently, by the dy-namics of the disease. For example, TNF-a–encoding DNAvaccines could be used as a practical way of combating RA.However, it might become problematic if the subject undergoespregnancy. Because CaP is a terminal disease in men, and becausemice lacking CCL2 display minimal side effects, this drawback islimited. 2) Previous studies, by several groups, including ours,showed that in the absence of anti-self–Abs, DNA vaccines mightgenerate an opposing effect. That is, without Ab-based neutrali-zation the gene product of the vaccines would be functional andprovoke an effect opposing the one desired by the vaccine (47–49). This strategy has been successfully implemented for thera-peutic target expression of gene products at autoimmune sites (47–49). Thus, potentially, it is possible that in some patients who haveCaP, CCL2-encoding vaccine would induce an opposing effect. Asimple way to avoid this difficulty could be to use a plasmid DNAencoding an N-terminal truncated CCL2, in which the geneproduct is inactive (50).The reason for selecting the C1-induced disease as a model to

investigate the validity of our findings is that in this model, as withthe human disease, CCL2 dominates the chemokine expression atthe tumor site. C1 very effectively forms tumors in immuno-competent mice, whereas C3 lacks this ability as shown by others(31) and verified in this study (Fig. 1A). Subsequently, of theselines only C1 led to an increased transcription of CCL2 at thetumor site (Fig. 2B). This could result not only from direct pro-duction of this chemokine by the tumor cells, but also from anupregulation of CCL2 transcription by macrophages and by thestroma cells that support the tumor (51). Nevertheless, the con-sequence of this dominant transcription is the selective loss oftolerance to CCL2 leading to the development of CCL2-specificauto-Abs (Fig. 2C) that continue to persist along the developmentof the tumor (Fig. 2D), and as neutralizing Abs (Fig. 2E) possiblyparticipate in its regulation. By showing that targeted amplifica-tion of their function suppress tumor development, we suggest anapplicative way of therapy that could also be explored in humans.In an attempt to further validate our findings, we investigated

selectivity of tolerance breakdown in TRAMP mice due to over-expression of SV40 develop CaP (24). Surprisingly, we found that,even though the C1 line is derived from TRAMP mice (24), inthese mice the expression of the CCR5 ligands CCL3 and CCL5 atthe primary tumor overrides CCL2 that is also apparently ex-pressed, although to a lower relative level (not shown). Sub-sequently, these mice generate selective auto-Ab titer to thesechemokines over CCL2. These results further support our workinghypothesis that selective breakdown of immunological tolerancein humans and mice is directed by dominantly expressed in-

flammatory mediators at an immune-restricted site undergoinga destructive process. CCR5 ligands have been recently suggestedto serve as growth/survival factors for androgen-dependent cancerdiseases, including CaP (52, 53). In future studies, we intend to

use this model and the DNA vaccination technology to furtherinvestigate their role in CaP.The other applicative outcome of our study also refers to the

diagnosis of the malignant stage of disease. The current biomarker

commonly used for the diagnosis of CaP is sera levels of prostate-specific Ags (PSAs) that are elevated in accordance with hyper-trophy of the prostate gland and cannot predict transition to ma-lignancy (54, 55). Another limitation of measuring PSAs asa major screening tool for the diagnosis of CaP is that significant

portions of malignant subjects (∼15%) have low levels of PSAswith low velocity (56). It remains to be examined how disease-specific is the production of CCL2-specific Abs. Yet, it could serveas an important complementary or even alternative biomarker thatwould not only identify the transformation from benign hyper-

trophy to malignancy, but also identify low levels of PSAs insubjects with developing CaP.There is an open-ended question about the in-depth mechanistic

basis of immunological tolerance breakdown under the conditionsin which inflammatory mediators are predominantly expressed at

a restricted tumor or autoimmune site undergoing intensive de-struction. We think that natural adjuvants relapsed from destructeddying cells provide the appropriate environment for tolerancebreakdown (57). Future studies will elaborate this hypothesis.Finally, it should be noted that effective neutralization of CCL2

slows tumor development, but does not eradicate it. Thus, it islikely that other factors are involved in containing the tumor. Thissuggests that an effective therapy for CaP should combine in-hibition of a keymediator that supports tumor development, such as

CCL2, together with other ways of therapy, as was recentlydemonstrated in a study that used anti-CCL2 and docetaxel as aneffective combined therapy (13).

DisclosuresN.K. and G.W. hold pending patents on the therapy of autoimmunity and

cancer using CCL2-encoding DNA vaccines, and on using anti-CCL2 Abs

for the diagnosis of prostate cancer.

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The Journal of Immunology 1101

by guest on June 18, 2018http://w

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.jimm

unol.org/D

ownloaded from

Corrections

Izhak, L, G.Wildbaum,W. Uri, Y. Shaked, J. Alami, D. Dumont, A. Stein, and N. Karin. 2010. Predominant expression of CCL2 at the tumorsite of prostate cancer patients directs a selective loss of immunological tolerance to CCL2 that could be amplified in a beneficial manner.J. Immunol. 184: 1092–1101.

The seventh author’s name was omitted from the article. In addition, the third author’s name was published incorrectly. The correctedauthor and affiliation lines are shown below.

Liat Izhak,* Gizi Wildbaum,* Uri Weinberg,* Yuval Shaked,† Jennifer Alami,‡ Daniel Dumont,‡ Boris Friedman,x Avi Stein,x andNathan Karin*

*Department of Immunology and †Department of Pharmacology, the Ruth and Bruce Rappaport Faculty of Medicine, RappaportFamily Institute for Research in the Medical Sciences, Technion-Institute of Technology; xDepartment of Urology, Carmel Medical Centerand the Ruth and Bruce Rappaport Faculty of Medicine, Haifa, Israel; and ‡Department of Medical Biophysics, University of Toronto,Toronto, Ontario, Canada

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1090025

Copyright � 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

The Journal of Immunology

Corrections

Izhak, L., G. Wildbaum, W. Uri, Y. Shaked, J. Alami, D. Dumont, A. Stein, and N. Karin. 2010. Predominant expression of CCL2 at thetumor site of prostate cancer patients directs a selective loss of immunological tolerance to CCL2 that could be amplified in a beneficialmanner. J. Immunol. 184: 1092–1101.

Due to a technical mistake, the image for Fig. 3Cf was used in both Fig. 3Ce and Fig. 3Cf. The corrected Fig. 3 in which the image forFig. 3Ce has been replaced is shown below. The figure legend was correct as published and is shown below for reference.

Copyright � 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

The Journal of Immunology 2509

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800167

FIGURE 3. CCL2-encoding plasmid DNA

amplifies auto-Ab production to CCL2 and

suppresses the development of the primary

tumor. A, The kinetics of CCL2-specific

auto-Ab titer developed in groups of mice in-

jected with 7 3 106 C1 cells/mouse s.c.,

which were or were not injected 25 d later,

to repeated injections (100 mg DNA in PBS,

every 5 d) of pcDNA-CCL2, pcDNA-b–actin

or PBS, compared with naive mice. Results

are shown as mean log2 Ab titer 6 SE of six

mice per group. B, Mice of the above groups

were monitored for the growth of the primary

tumor every 5 d by an observer blind to the

experimental protocol. Results of one of three

experiments, with similar data, are shown as

mean six mice 6 SE. Tumor development in

pcDNA-CCL2–vaccinated mice was signifi-

cantly reduced (p , 0.001). C, On day 70,

the mice were euthanized and subjected to

immunofluorescence and immunohistochemi-

cal analyses using anti-CD31 (a–c, frozen

sections), anti-F4/80 (d–f), and anti-VEGF

(g–i) (original magnification 340).

2510 CORRECTIONS