Maximal T Cell Mediated Antitumor Responses Rely upon …Microenvironment and Immunology Maximal T Cell–Mediated Antitumor Responses Rely upon CCR5 Expression in Both CD4þ and CD8þ

Microenvironment and Immunology

Maximal T Cell–Mediated Antitumor Responses Rely uponCCR5 Expression in Both CD4þ and CD8þ T Cells

Alicia Gonz�alez-Martín1, Lucio G�omez1, Joseph Lustgarten2,†, Emilia Mira1, and Santos Mañes1

AbstractImmune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemo-

kines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5),exerts major regulatory effects on CD4þ- and CD8þ T cell-mediated immunity. Although CCR5 and its ligandsparticipate in the response to various pathogens, its relevance to tumoral immune control has been debated.Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptivetransfer studies, efficient tumor rejection required CCR5 expression by both CD4þ and CD8þ T cells. CCR5activation in CD4þ cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cellsand enhanced CD8þ T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcomaincidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenicTg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses inthese mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependentCD8þ T-cell activation. Cancer Res; 71(16); 5455–66. �2011 AACR.

Introduction

For decades, immunologists have tried to exploit the spe-cificity of the immune system to discriminate between normaland malignant tissue for cancer therapy. Success with certainimmunotherapeutic approaches shows that the immune sys-tem can restrict the onset and/or progression of some malig-nancies (1–3). Complete, durable antitumor responses arenonetheless rare, due largely to the induction of a regulatoryenvironment in which immune tolerance dominates overactivation (4). The challenge for immunotherapy is thus toidentify cell networks and molecular factors that enhance theexpansion and activation of tumor-reactive effector T cells.Immune responses are multistep processes that entail

antigen presentation, optimal triggering of specific T cells,and localization of immune effectors to appropriate sites.Completion of these steps requires intricate leukocyte traf-ficking patterns coordinated by chemokines and their recep-tors (5). CCR5 and its ligands CCL3, CCL4, and CCL5 have

emerged as key regulators of T-cell function. CCR5 is a centralelement in modulating helper-dependent CD8þ T-cellresponses, by guiding these cells to productive CD4þ/anti-gen-presenting cell (APC) complexes (6). CCL5 produced bytumor-infiltrating CD4þ T cells is also involved in recruitingAPC to the tumor site where, after CD40-mediated activation,they become competent to crosspresent and trigger CD8þ Tcells in the tumor parenchyma (7). In vitro studies indicatedthat CCR5 might also have migration-independent effects onT-cell activation, by participating in the costimulation ofCD4þ lymphocytes in cooperation with the TCR (8–10). Itremains unclear whether CCR5 effects on T-cell function arerelevant in physiopathologic situations, or are restricted tocertain conditions of immune activation. Moreover, to ourknowledge, no studies have addressed the precise role of CCR5in in vivo CD4þ T-cell function. For full comprehension of therole of CCR5 in T cell-mediated responses, it is particularlyimportant to determine whether its expression in CD4þ

lymphocytes is necessary for optimal dendritic cell (DC)activation and antigen crosspresentation.

Although CCR5 participates in the response to variouspathogens in mice and humans (11, 12), the relevance of thisreceptor and its ligands in the immune control of tumors isdebated. Elevated serum CCL5 levels are associated withbreast and cervical cancer progression (13). Evidence alsoimplicates CCR5 in the induction of proliferation, metastasis,and angiogenesis (14, 15), supporting its protumorigenic role.Some of these activities have been linked to the ability of CCR5to recruit macrophages and other immune suppressor cells tothe tumor site, creating an inflammatory environment thatpromotes tumor progression and immune evasion (16–19). Incontrast, increased levels of CCR5 ligands improved antitumorresponses in mice (20–25) and in humans (26). Furthermore,

Authors' Affiliations: 1Department of Immunology and Oncology, CentroNacional de Biotecnología/CSIC, Madrid, Spain; and 2Department of Immu-nology, Cancer Center Scottsdale, Mayo Clinic Arizona, Scottsdale, Arizona

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).† Dedicated to the memory of our collaborator Dr. Joseph Lustgarten, whodied on June 30, 2011.

Corresponding Author: Santos Mañes, Department of Immunology andOncology, Centro Nacional de Biotecnología/CSIC, Darwin 3, CampusCantoblanco, Madrid, Spain. Phone: 34-91-585-4840; Fax: 34-91-372-0493; E-mail: [email protected] or Emilia Mira, Phone: 34-91-585-4655; Fax: 34-91-372-0493; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1687

�2011 American Association for Cancer Research.

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the ccr5D32 polymorphism, which causes defective CCR5expression in humans, is associated with poor prognosis incolorectal (27) and breast (28) cancers, and with reducedefficacy of immunotherapy in melanoma (29).

Here, we provide genetic evidence for a CCR5 requirementto maximize the immune response to tumors. Our data show apreviously unreported role for CCR5 in optimizing CD4þ T-cell help in vivo, with clinical implications in the design oftherapeutic intervention for cancer patients.

Materials and Methods

MiceC57BL/6J wild-type (WT), CCR5�/�, CCR1�/�, PL-Thy1a/CyJ

(Thy1.1), Tg(TcraTcrb)1100Mjb/J (OT-I), Tg(TcraTcrb)425Cbn/J (OT-II), and FVB/N-Tg(MMTVneu)202Mul/J (MMTV-neu)mice were from The Jackson Laboratory and C57BL/6-Rag2�/�mice from Taconic. OT-I and OT-II mice were crossedwith CCR5�/� mice and bred to homozygosis for CCR5.CCR5�/� mice were crossed with mouse mammary tumorvirus (MMTV)-neu mice to generate MMTV-neu-CCR5þ/�

and backcrossed for 10 generations on the FVB background[�99.6% FVB, determined by speed congenics analysis by using220DNAmicrosatellitemarkers (Bionostra)]maintainingCCR5in heterozygosis. Live animal experiments were supervised bythe Centro Nacional de Biotecnología Ethics Committeeaccording to national and European Union guidelines.

Tumor cell linesTheC57BL/6 syngeneic tumor cell lines EG7, LLC [both from

the American Type Culture Collection (ATCC)] and Panc02were cultured in RPMI or Dulbecco's modified Eagle's mediumwith 10% FBS, 2 mmol/L L-glutamine and antibiotics; b-mer-captoethanol (50 mmol/L) was added to EG7 cultures. Panc02-luc cells were generated by transduction with pRV-IRES-LUCretroviruses. Firefly luciferase was amplified from the pGL3-basic vector (Promega) using 50-CGATTTAAATCCACC-ATGGAAGACGCC-30 and 50-GCGCGGCCGCTTACACGGC-GATCTTCCG-30, and subcloned in SwaI/NotI-digested pRV-IRES (Genetrix). Retroviruseswere generated by cotransfectionof 293T cells with pRV-IRES-LUC, pML-GAG-POL, and pVSV-Gplasmids (Genetrix) and used to infect Panc02 in the presenceof polybrene (4mg; Sigma). Transduced cells were subcloned bylimiting dilution and selected by Luc expression.

LLC-CCL5 cells were obtained by transduction withretroviruses generated by cloning CCL5 cDNA into BamHI/XhoI-digested pRV-IRES-LUC plasmid. Short hairpin RNA(shRNA) for murine CCL5, shRNA-CCL5 pSM2026-C-3 (cloneV2MM_73846, Open Biosystems), was packaged in retroviralparticles and used to generate LLC-si-CCL5 cells. Stable LLC-CCL5 cells were obtained by limiting dilution, and LLC-si-CCL5 cells were selected with puromycin (2 mg/mL).

Concentrations of endogenous or ectopically expressedCCL3,CCL4, and CCL5 were determined by ELISA (R&D; Biosource).

Quantitative RT-PCR analysescDNA was synthesized by reverse transcription (Promega)

of total RNA (1 mg). Quantitative reverse transcriptase PCR

(qRT-PCR) reactions were done with the Power SYBR GreenPCR Master Mix System (Applied Biosystems) with specificprimers for mouse CCR1, CCR3, and CCR5 using b-actin tonormalize data.

Tumor inductionSyngeneic tumors were induced by injection of Panc02-Luc

(5 � 106), LLC sublines (5 � 105), or EG7 (5 � 106) cells inindicated hosts. For chemical carcinogenesis, mice wereinoculated (s.c.) with a single dose of 3-methylcholanthrene(MCA; 100 mg; Sigma) dissolved in corn oil (Sigma). Breasttumors in MMTV-neu mice were detected by weekly palpa-tion.

In all cases, tumors were measured periodically with cali-pers, and volume calculated (length � width2/2). Tumorgrowth was analyzed by bioluminescence after inoculation(intraperitoneally) of mice with D-luciferin (15 mg/g); imageswere recorded with a DCC camera (Hamamatsu) and signalemission quantified with Wasabi software. At the endpoint,tumors were disaggregated to obtain single cell suspensionsand analyzed for immune cell infiltration by staining withanti-CD4, -CD45, -Gr1 (Beckman Coulter), -CD11c, -CD19,-Mac3, -NK1.1, -CD3, -CD8, and -CD11b (eBioscience) forFACS (FACS Calibur, Beckton Dickinson).

Adoptive transfer experimentsWT and CCR5-deficient OT-I and OT-II cells were purified

by negative selection [Dynal Mouse CD8 and CD4 NegativeIsolation Kits (Invitrogen), respectively]. Combinations ofthese cells (5 � 106 each) were adoptively transferred intoCCR5�/� or Thy1.1 mice bearing EG7 tumors [a thymiclymphoma stably expressing ovalbumin (OVA) protein; ATCC]generated 3 days earlier by s.c. inoculation (5� 106 cells) in theright flank. Tumor volume was calculated as above.

Homing assaysThy1.1 mice bearing EG7 tumors received OT-I and OT-II

cells by adoptive transfer. After 4 and 7 days, cell suspensionsfrom tumors, draining lymph nodes (dLN), and non-dLN werestained with anti-Thy1.2 (clone 53-2.1), -Va2 (B20.1), -Vb5-biotin (MR9-4; BD-Pharmingen), and -CD8 (53-6.7) or -CD4(RM4-5; eBioscience), followed by streptavidin-APC (BeckmanCoulter), and were analyzed by FACS (BD LSRII System;Becton Dickinson). OT-I and OT-II cells were identified asThy1.2þVa2þVb5þCD8þ and Thy1.2þVa2þVb5þCD4þ,respectively.

Activation assaysFor in vivo assays, Thy1.1 mice bearing EG7 tumors received

OT-IþOT-II cells. After 6 days, cell suspensions from tumorsand dLN were assayed for IFNg production. Tumor lympho-cytes were partially purified by Ficoll–Hypaque from a pool of4 tumors per condition. Lymphocytes (5 � 106) from eachcompartment were cocultured (5 hours, 37�C) with OVA (257–264) þ OVA (323–339; 1 and 10 mg/mL, respectively)-pulsedThy1.1-splenocytes (2 � 106; 1 hour, 37�C) in the presence ofbrefeldin A (Sigma). IFNg production in OT-I cells was deter-mined by fluorescence-activated cell sorting (FACS); cells were

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stained with anti-Thy1.2-FITC (53-2.1) and -CD8-PE (53-5.8;BD-Pharmingen), then permeabilized (Cytofix/Cytoperm kit;BD-Pharmingen) and stained with anti-IFNg (XMG1.2; BD-Pharmingen).For CD69 determination, dLN cell suspensions were stained

with anti-Va2 (B20.1), -CD4 (RM4-5), -Vb5-biotin (MR9-4; BD-Pharmingen), -CD69 (H1.2-F3), and -CD8 (53-6.7; eBioscience),followed by streptavidin-APC and analyzed by FACS (BDLSRII).For CD40L determination, bone marrow–derived dendritic

cells (BMDC) were prepared from bone marrow cells culturedwith murine granulocyte-macrophage colony-stimulating fac-tor (15 ng/mL) and IL-4 (10 ng/mL); at day 7 these cellsexpressed CD11c, CD80, CD86, and MHC-II, as determined byFACS (not shown). BMDC were pulsed with OVA (323–339; 10mg/mL) or medium (unpulsed) in the presence of LPS (20 ng/mL; 1 hour, 37�C), and then cocultured (48 hours) withpurified OT-IIWT or OT-IIKO cells. CD40 and CD40L weredetected by staining with anti-CD40 (3/23), -CD11c (HL3),-CD40L-biotin (MR1; BD-Pharmingen), -CD3 (145-2C11) and-CD4 (RM4-5; eBioscience), followed by streptavidin-eFluor450 (eBioscience). CD11cþ cells were stained withanti-I-A/I-E (2G9; MHC-II), -CD80 (16-10A1), -CD86-biotin(GL1; BD-Pharmingen) and -CD11c (N418; eBioscience), fol-lowed by streptavidin-eFluor450. IFNg production in OT-IIcells was determined by staining with anti-CD3 (145-2C11),-CD4 (GK1.5; Beckman Coulter) and -IFNg (XMG1.2; BD-Pharmingen) in brefeldin A-treated cells. Samples were ana-lyzed by FACS (GALLIOS; Beckman Coulter).For in vitro assays, OT-I and OT-II cells (2 � 105 each) were

cocultured (20 hours, 37�C) with OVA peptide-pulsed spleno-cytes (106) as above; brefeldin A was added for the last 5 hoursof incubation. IFNg production in OT-I cells was assessed byFACS as above.

Immunization assaysCCR5�/�mice received adoptive transfers of OT-I and OT-II

cells (3 � 106 each); after 24 hours, they were immunized by s.c. injection in the hind foot with alum mixed with OVA (323–339; 10 mg) and CpG (GCTAGACGTTAGGT andTCAACGTTGA; 20 mg total). OT-I cells were labeled withCFSE (5 mmol/L, 10 minutes, 37�C; Invitrogen) prior to trans-fer. OT-I cells in the dLN and the contralateral popliteal LNwere quantified by FACS after 40 hours.

Chemokine production in dLNCCR5�/� mice transferred with OT-IIWT and OT-IIKO cells

(3 � 106) were immunized with OVA (323–339) peptide asabove. dLN were extracted after 30 hours and incubated (12hours) in RPMI medium (50 mL) containing 10% FCS. CCL3,CCL4, and CCL5 in conditioned media were quantified byELISA (R&D Systems).

CpG treatment of tumors in Tg-neu miceSpontaneous breast tumors in MMTV-neu and MMTV-neu-

CCR5�/� mice were detected by palpation. Tumor-free micewere inoculated s.c. in the right flank with 5 � 105 N202.1Acells (30). These cells express high Her-2/neu levels and form

tumors histologically similar to spontaneous tumors inMMTV-neu but not in conventional FVB mice. Intratumorinjections of CpG (ODN-1826, InvivoGen; 30 mg/mouse,3 times/week) were initiated 16 days after tumor inoculation(tumor volume �100 mm3); tumor volume was measuredtwice weekly. After sacrifice of mice, cell suspensions fromtumors and dLN were stained for neu-specific T cells withH2Dq/rat-neu (420–429) tetramer (National Institute ofAllergy and Infectious Diseases, Tetramer Core Facility,Emory University, Atlanta, GA). Cells from tumors anddLN were stained with anti-CD25 (7D4; BD-Pharmingen),-FoxP3 (FJK-16s), and -CD4 (GK1.5; eBioscience) to detectregulatory T (Treg) cells. Splenocytes (5 � 106) were resti-mulated ex vivo (22 hours) with rat-neu (420–429) peptide(10 mg/mL), and secreted IFNg was quantified by Luminex(Biosource).

Statistical analysisComparison of 2 data groups was done with the 2-tailed

Student's t test, using logit transformation for percentages.Dunnett or Kruskal–Wallis tests were used for multiple com-parisons. The log-rank test was used to compare Kaplan–Meier curves.

Results

CCR5 expression in the host delays tumor growthWe determined the growth of s.c. grafted tumors in syn-

geneic, immunocompetent WT, and CCR5�/� mice. To avoidpossible cell-autonomous effects, we used the Lewis lungadenocarcinoma (LLC) and a luciferase-expressing variantof the pancreatic adenocarcinoma Panc02 (Panc02-Luc), asno mRNA encoding CCR5 was detected in these cell lines(Supplementary Fig. S1A). LLC and Panc02-Luc cellsexpressed high levels of CCR1, which shares binding withCCR5 to CCL3 and CCL5, and marginal levels of CCR3, whichalso binds CCL5 (Supplementary Fig. S1A). LLC produced lowlevels of CCL5, whereas Panc02-Luc cells produced CCL4 andCCL5 (Supplementary Fig. S1B).

Tumors formed by LLC cells grew more rapidly (Fig. 1A)and were ultimately larger (Fig. 1B) in CCR5�/� than in WTrecipients. These CCR5-dependent differences were exa-cerbated for tumors induced with Panc02-Luc cells(Fig. 1C). Reduced Panc02-Luc tumor growth in WT micewas confirmed by bioluminiscence (Fig. 1D and E). Theseresults indicate that host CCR5 expression restricts tumorgrowth.

The differences in tumor growth were not associated withchanges in the frequency of proliferating (phospho-histoneH3þ) or apoptotic (TUNELþ) cells between WT and CCR5�/�

hosts (not shown). There were nonetheless statistically sig-nificant changes in the pattern of leukocytes infiltratingPanc02-Luc–derived tumors. CCR5 is expressed in CD4þ

and CD8þ lymphocytes, NK and NKT cells, monocytes, macro-phages, and immature DC. FACS analyses showed greaterinfiltration by DC and T cells, particularly CD8þ cells, intumors growing in WT than in CCR5�/� hosts (Fig. 1F);infiltrating myeloid cells showed the opposite pattern. We

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detected no CCR5-dependent changes in natural T regulatory(Treg) cells infiltrating LLC or Panc02-Luc tumors (Fig. 1G).NK cells could not be identified reliably in these tumors.

CCR5, but not CCR1, restricts tumor growth in anagonist-dependent manner in immunocompetent hosts

Host-associated differences in LLC- and Panc02-Luc tumorgrowth might be explained by the distinct CCR5 agonist levelsin these cell lines (Supplementary Fig. S1B). We generated LLCcell lines in which CCL5 secretion was enhanced approxi-mately 20-fold (LLC-CCL5) or decreased approximately 4-fold(LLC-siCCL5) compared with the parental cell line (LLC-mock; Supplementary Fig. S2). LLC-mock, -siCCL5, and-CCL5 cells proliferated in vitro at similar rates (not shown).LLC-siCCL5 formed larger tumors than LLC-mock cells,whereas LLC-CCL5 cells formed the smallest tumors when

injected into WT but not into CCR5�/� mice (Fig. 2A). Wefound no changes in the frequency of proliferating or apop-totic tumor cells, or in blood vessel number and diameter as aresult of CCL5 overexpression or silencing (SupplementaryFig. S3).

Because CCL5 is also a CCR1 agonist, we analyzed thegrowth of tumors formed by these LLC sublines in CCR1�/�

mice. LLC-siCCL5 cells again formed the largest and LLC-CCL5 cells formed the smallest tumors in CCR1�/� recipients(Fig. 2B). There were no differences in the final volume oftumors formed by LLC-mock, -CCL5, or -siCCL5 cells inocu-lated in parallel inWT and CCR1�/�mice (Fig. 2C). The resultsindicate that CCR5 specifically restricts tumor growth in anagonist-dependent manner.

Because CCR5 expression influences T-cell infiltrationinto Panc02-Luc tumors (Fig. 1F), we tested whether CCL5

Figure 1. Tumor growth isinhibited in CCR5�/� mice. A, LLCtumor growth in WT and CCR5�/�

mice. Each data point is the mean� SEM of tumor volume of 1representative experiment of 3conducted experiments (n ¼ 4mice/group). B, weight of LLCtumors in (A) at day 21. C, growthkinetics of Panc02-Luc tumorgrafts in WT and CCR5�/� mice(n ¼ 5 mice/group). D,bioluminescence images ofPanc02-Luc tumor-bearing micein (C) at day 7 postcell inoculation.Luciferase activity was measuredand transformed to a pseudocolorimage; red represents maximallight intensity. E, quantification ofthe relative bioluminescent signal(RLU) in (D). F, FACS analysis ofthe infiltrate in Panc02-Luc tumorsin WT and CCR5�/� mice (day 22postinoculation). G, specificstaining of Treg(CD4þCD25þFoxP3þ) cells in LLCand Panc02-Luc tumors.*, P < 0.05, 2-tailed Student'st test.






overexpression restricted tumor growth in RAG2�/� mice,which lack mature B and T lymphocytes. Growth kinetics wassimilar for LLC-mock, -CCL5, and -siCCL5 tumors induced inRAG2�/�mice (Fig. 2D). These findings implicate the adaptiveimmune system as a mechanism by which CCR5 restrictstumor growth.

CCR5 expression in T cells is necessary for optimalantitumor responsesTo determine how CCR5 regulates T-cell antitumor

responses, we crossed CCR5�/� mice with OVA-specificOT-I (CD8þ) or OT-II (CD4þ) transgenic mice. As tumor cells,

we used the EG7 thymoma, which does not express CCR5mRNA (not shown), but secretes the CCR5 ligands CCL3(�120 pg/mL/24 hours) and CCL4 (�15 pg/mL/24 hours).Initial experiments showed more rapid EG7 tumor growth inCCR5�/� than inWTmice (Supplementary Fig. S4), suggestinga CCR5-mediated polyclonal response in WT mice. Becausepolyclonal CD8þ T cells enhance the response of antigen (Ag)-specific cytotoxic T lymphocytes (CTL), a CCR5-dependentphenomenon termed CD8þ T-cell help (31), we used CCR5�/�

mice as tumor hosts in subsequent experiments.Adoptive transfer of single WT (OT-IWT or OT-IIWT) or

CCR5-deficient CD4þ (OT-IIKO) or CD8þ (OT-IKO) T cellspoorly restricted growth of EG7-induced tumors (Supplemen-tary Fig. S5A and B). Optimal tumoricidal activity usuallyinvolves cooperation between CD4þ and CD8þ T cells (32);this cooperation was evident in mice coinjected with OT-IWT

plus OT-IIWT cells, which led to greater restriction of tumorgrowth than in mice coinjected with OT-IKOþOT-IIKO mix-tures (Fig. 3A). Tumor rejection was complete in 9/13 OT-IWTþOT-IIWT cell recipients but in only 2/15 OT-IKOþOT-IIKO

recipients. These results suggest CCR5-mediated, CD4þ/CD8þ

cooperation for optimal T cell-mediated elimination of EG7tumors.

CCR5 enhances tumor-specific CD8þ T lymphocyteinfiltration and effector function

To determine the effect of CCR5 expression on T-cellinfiltration into the tumor parenchyma, we injected OT-IWTþOT-IIWT or OT-IKOþOT-IIKO cell mixtures into EG7-bearing mice, and used FACS to identify transferred cellsinfiltrating the tumor days 4 and 7. At day 4, transferredOT-I or OT-II cells were undetectable. At day 7, we observed anincrease in tumor-infiltrating OT-IWT versus OT-IKO cells(Fig. 3B); OT-IIWT and OT-IIKO cells were undetectable intumors. There was no difference in OT-I and OT-II cellnumbers in dLN (Supplementary Fig. S6) at day 7, indicatingthat CCR5 does not affect T-cell trafficking to LN.

The activation state of CD8þ T cells, which can be mon-itored by IFNg production, influences their infiltration intotumors (33, 34). The percentage of IFNg-producing OT-I cellsand mean fluorescence intensity (MFI) of IFNg staining wereincreased in tumors (Fig. 3C and D) and in dLN (Fig. 3E and F)from OT-IWTþOT-IIWT compared with OT-IKOþOT-IIKO cellrecipients. OT-IWT cells also tended to express higher levels ofthe CD69 activation marker than OT-IKO cells (Fig. 3G),although differences were not significant; CD69 expressionwas similar in OT-IIWT and OT-IIKO cells (Fig. 3G).

CCR5 expression in CD4þ T cells is needed for maximalCD8þ T-cell tumoricidal activity

CD4þ/CD8þ T-cell help requires CCR5 expression by CD8þ

T cells (6). To test whether this cooperation also requiresCCR5 expression in CD4þ cells, we analyzed the efficiency ofEG7 tumor rejection following OT-IWTþOT-IIKO or OT-IKOþOT-IIWT cell transfer. Lack of CCR5 expression in eitherOT-I or OT-II cells resulted in inefficient restriction of tumorgrowth compared with control OT-IWTþOT-IIWT cell recipi-ents (Fig. 4A). Growth kinetics correlated with the number of

Figure 2. CCL5 levels regulate tumor growth in immunocompetent hostsin a CCR5-dependent manner. A, growth kinetics of LLC tumors withforced or silencedCCL5 expression inWT andCCR5�/�mice. B, growth ofLLC cell lines as above in CCR1�/� mice. C, final volume of LLC tumorswith forced or silenced CCL5 expression in WT and CCR1�/� mice. D,growth of tumors induced with the same LLC lines in RAG2�/� mice. In allcases, n ¼ 5 mice/group. One representative experiment of 2 is shown(except for CCR1�/� mice).






tumor-infiltrating OT-I cells, which was maximal in OT-IWTþOT-IIWT cell recipients (Fig. 4B). These data suggestedthat CCR5 expression in OT-II cells regulates OT-I cell traf-ficking into the tumor mass.

To test whether CCR5 expression in OT-II cells influencedOT-IWT cell activation, we stained CTL isolated from a pool of4 EG7 tumors from OT-IWTþOT-IIWT or OT-IWTþOT-IIKO cellrecipients with anti-IFNg antibodies. The percentage of IFNgþ

OT-I cells and MFI values were increased in tumors from OT-IWTþOT-IIWT compared with OT-IWTþOT-IIKO cell recipients(Fig. 4C). OT-I cell activation was likewise higher in dLN fromOT-IIWT than from OT-IIKO cell-transferred mice (Fig. 4D andE). CCR5 expression on CD4þ cells could thus be an importantfactor for CD4þ T-cell help in CD8þ T-cell activation.

CCR5 enhances CD40L levels in antigen-stimulatedCD4þ T cells and APC maturation

Ag-specific interaction of CD4þ cells with APC in LNinduces secretion of the proinflammatory chemokinesCCL2, CCL3, and CCL4 (6, 35). CCL3 and CCL4 producedby CD4þ T cells and APC attract CD8þ T cells to productivecrosspriming (6). We observed CCL3, CCL4, and CCL5 pro-duction in dLN of OT-IIWT or OT-IIKO cell-transferred miceimmunized in the hind foot with the OVA (323–339) peptide,

the cognate antigen (Ag for OT-II cells). CCL3 and CCL5 levelswere significantly higher in dLN from OT-IIWT compared withOT-IIKO cell recipients (Fig. 5A); in vitro analyses showed thatthese chemokines were produced by APC and CD4þ T cells(Supplementary Fig. S7). Despite the increase in chemokineproduction, there was no difference in OT-I cell recruitment indLN fromOVA (323–339)-immunized OT-IWTþOT-IIKO or OT-IKOþOT-IIWT cell recipients compared with controls (Fig. 5B);there was nevertheless a significant reduction in CD8þ T-cellrecruitment to dLN from OT-IKOþOT-IIKO cell-transferredmice (Fig. 5B). These findings confirm a role for CCR5 inactive CD8þ T-cell recruitment to dLN, although CCR5 depen-dency was apparent only when the receptor was absent inboth CD4þ and CD8þ T cells.

The CD4þ T-cell help mechanism for CD8þ T-cell activationalso involves APC conditioning via binding of CD40 on theAPC to CD40L on the CD4þ T cell (34). CD40L is upregulatedby TCR engagement, which also induces autocrine secretion ofCCR5 ligands (Refs. 8, 9; and not shown). We found that CD40Llevels were higher in WT than in CCR5-deficient CD4þ Tcells following anti-CD3–mediated activation (Fig. 5C).CD40L upregulation was also higher in OT-IIWT than inOT-IIKO cells cocultured with OVA (323–339)-loaded BMDC(Fig. 5D); as predicted (8), CCR5 expression also enhanced

Figure 3. CCR5 expression in T cells is necessary for optimal antitumor responses. A, growth of EG7 grafts after adoptive transfer (day 0) of OT-IWTþOT-IIWT

or OT-IKOþOT-IIKO cells. Each data point is the mean� SEM of tumor volume (n¼ 13 and 15mice, respectively). B, number of OT-I cells (Thy1.2þ/Va2þ/Vb5þ/CD8þ) infiltrating EG7 tumors implanted in Thy1.1þ recipients adoptively transferred with OT-IWTþOT-IIWT or OT-IKOþOT-IIKO cells (n ¼ 9 mice/group;*, P ¼ 0.006). C and D, cell suspensions from 4 EG7 tumors at day 6 posttransfer were restimulated ex vivo with OVA (257–264) peptide, and lymphocytesstained with anti-IFNg and -Thy1.2 antibodies. Representative dot plots (in the CD8þ gated population) are shown (C); (D) shows mean � SD of thefrequency of IFNgþOT-I cells (left) and theMFI of staining (right) from 2 independent experiments (*, P¼ 0.02). E and F, dLN cell suspensions were restimulatedas in (C) and stained to detect IFNg . Representative dot plot (E) and individual data points (F) for the percentage of IFNgþOT-I cells (*, P¼ 0.049) andMFI values(*, P ¼ 0.04; n ¼ 11 mice/group). G, percentage of CD69þCD4þ and CD69þCD8þ cells in dLN isolated in (E).






OT-II activation, as determined by IFNg production (Fig. 5E).Direct CCL4 stimulation of CD4þ T cells nonetheless did notinduce CD40L expression (not shown), suggesting that CCR5acts as a costimulator for TCR-mediated CD40L induction.We tested whether increased CD40L levels in CCR5-expres-

sing CD4þ T cells affect DC maturation and subsequent CD8þ

T-cell activation. Levels of MHC class II (Fig. 5F), CD80(Fig. 5G), and CD86 markers (Fig. 5H) were higher in BMDCcocultured with OT-IIWT than with OT-IIKO cells; CD40 expres-sion on the BMDC was similar and independent of the CCR5genotype of CD4þ T cells (Supplementary Fig. S8).Finally, we analyzed whether CCR5 expression on CD4þ T

cells affects CD8þ T-cell crosspriming. APC were incubatedwith class I- and class II-restricted OVA peptides and thencocultured with OT-IIWT or OT-IIKO and OT-IWT cells. IFNgproduction was significantly increased by OT-I cells whencocultured with OT-IIWT-APC compared with APC-OT-IIKO

cell complexes (Fig. 5I). The results suggest that CCR5 expres-sion in CD4þ T cells improves APC maturation and subse-quent CD8þ T-cell activation.

CCR5 deficiency accelerates the onset of some primarytumors in the mouseThe evidence from this study with transplantable tumors

suggests that CCR5 plays a role in the immune control oftumor outgrowth. We next analyzed whether CCR5 affects

MCA-induced fibrosarcoma, used to study innate and adap-tive immune surveillance in cancer (36). Sarcoma incidencewas higher in CCR5�/� than in WT mice (Fig. 6A); the mediantime for tumor detection in 50% of the animals was 22.5 and15 weeks for WT and CCR5�/� mice, respectively. Fibrosar-comas also tended to grow more rapidly in CCR5�/� micethan in WT mice (Fig. 6B).

To evaluate whether CCR5 affects the onset of other pri-mary tumors, we crossed CCR5�/� mice on the FVB/N back-ground with transgenic FVB/N-Tg(MMTVneu)202Mul/J(MMTV-neu) mice, which overexpress the rat neu protoon-cogene in mammary tissue and develop spontaneous breasttumors (37). We observed no differences in tumor incidence(median time: 268 and 276 days for MMTV-neu and MMTV-neu-CCR5�/� mice, respectively; Fig. 6C), in tumor progres-sion as determined by the growth kinetics slope (Fig. 6D), or infinal tumor weight (Fig. 6E). The results concur with the lackof association between the ccr5D32 polymorphism, whichrenders a nonfunctional CCR5 receptor, and the incidenceof breast cancer in humans (28).

CCR5 enhances TLR-induced reactivation of antitumorimmune responses

Because CCR5 deficiency did not affect breast carcinomaonset in MMTV-neu mice, we used this model to analyzethe effect of CCR5 on immune-based therapies. Most

Figure 4. Lack of CCR5 expression in CD4þ T cells impairs CD8þ T-cell tumoricidal activity. A, tumor volume of EG7 grafts after adoptive transfer (day 0) of OT-IWTþOT-IIWT, OT-IWTþOT-IIKO, or OT-IKOþOT-IIWT cells (mean � SEM; n ¼ 10 mice/group). B, number of OT-I infiltrating EG7 tumors in Thy1.1þ

host mice receiving OT-IþOT-II mixtures as indicated (6 mice/group; *, P < 0.05 compared with control). C–E, mice bearing EG7 tumors were adoptivelytransferred with OT-IWTþOT-IIWT, or OT-IWTþOT-IIKO cells. Frequency of IFNgþOT-I cells and theMFI of IFNg staining was determined in lymphocytes isolatedfrom a pool of 4 EG7 tumors (C) and from individual dLN (D and E) after ex vivo restimulation with OVA (257–264) peptide. For dLN, representative dot plots areshown (D) with mean � SEM for the percentage of IFNgþ OT-I cells (left; *, P ¼ 0.034) and MFI values (right; *, P ¼ 0.023). E, n ¼ 4 mice/group.






spontaneous tumors induce suppression of tumor-experi-enced T cells by central and/or peripheral tolerancemechanisms (38). There is nonetheless a residual low avidity,tumor-associated antigen (TAA)-specific T cell repertoirethat, when stimulated, can elicit an effective antitumorresponse (39). MMTV-neu mice are functionally tolerantto neu antigens (40), but local or systemic administrationof TLR agonists can reactivate the antineu T-cell repertoireto restrict tumor growth (41).

We studied the reactivation of neu-specific T cells by theTLR9 agonist CpG (ODN-1826) in WT and MMTV-neu-CCR5�/� mice. To avoid ambiguity due to asynchronousappearance of spontaneous tumors, we generated graftswith the H2Dq/H2Lq-restricted N202.1A mammary cell linederived from an MMTV-neu mouse tumor. N202.1A cellsgrew similarly in untreated MMTV-neu and MMTV-neu-CCR5�/� mice (Supplementary Fig. S9). Intratumor injec-tion of CpG nonetheless induced a significant reduction intumor growth in MMTV-neu but not in MMTV-neu-CCR5�/� mice (Fig. 7A), suggesting that CCR5 expressionis necessary for TLR9-mediated stimulation of antitumorresponses.

Although CpG initially targets the innate immune system,CpG-induced responses are CD4þ and CD8þ T-cell depen-dent (41). We determined the number and activation state ofneu-specific CD8þ T cells using a tetramer loaded with therat-neu (420–429) peptide. Expansion of H2Dq/rat-neu (420–429) tetramer-positive cells was greater in dLN of CpGinjected than in control-injected MMTV-neu mice; thisenhancement was not observed in CpG-treated MMTV-

Figure 5. Increased CD40L levels in CCR5-expressing CD4þ T cells. A,CCL3, CCL4, and CCL5 levels in dLN of CCR5�/� mice adoptivelytransferred with OT-IIKO or OT-IIWT cells and immunized with OVA (323-339) peptide (n ¼ 4/6 per group; *, P ¼ 0.04, **, P ¼ 0.026). B, CCR5�/�

mice were adoptively transferred with the indicated OT-IþOT-II mixtures,then immunized with OVA (323–339) peptide. The number of OT-I cells wasdetermined in dLN and non-dLN and the ratio calculated. Individual datapoints and mean (line) are shown (*, P < 0.05). C, CD4þ T cells from WT orCCR5-deficient mice were seeded onto anti-CD3–coated plates andCD40L determined by FACS at indicated times (*, P ¼ 0.03; **, P ¼ 0.005).D–H, OT-IIWT or OT-IIKO cells were mixed with control (-Ag) or OVA (323–339)-loaded BMDC (þAg), and CD40L (D; *, P¼ 0.02), IFNg (E; *, P¼ 0.02),MHC-II (F; *, P ¼ 0.04), CD80 (G; *, P ¼ 0.03), and CD86 (H; *, P ¼ 0.05)levels were determined by FACS. I, OT-IWT and OT-IIWT or OT-IIKO cellswere mixed with OVA (323–339) plus OVA (257–264)-pulsed splenocytes;IFNg was determined by FACS in OT-I cells after 24 hours coculture. Datashow mean � SD from 1 representative experiment of 3 conductedexperiments.

Figure 6. Effect of CCR5 on carcinogen-induced and spontaneoustumor incidence. A, Kaplan–Meier analysis of fibrosarcoma-freeincidence comparing WT (n ¼ 18) and CCR5�/� (n ¼ 19) mice afterMCA injection (P ¼ 0.0038). B, growth kinetics of fibrosarcomas in WT(n ¼ 8) and CCR5�/� (n ¼ 16) mice. C, Kaplan–Meier plots show thepercentage of tumor free, virgin female MMTV-neu (n ¼ 268) and MMTV-neu-CCR5�/� (n ¼ 276) mice as a function of postnatal age (P ¼ 0.39).D, spontaneous tumor growth was analyzed for 6 weeks by weeklymeasurement. Tumor volume data for each tumor were logarithmicallytransformed and fitted by linear regression; slopes were then calculated.Data show mean � SEM of slopes for 33 and 11 tumors fromMMTV-neu and MMTV-neu-CCR5�/� mice, respectively. E, tumorweight from mice in (D).






neu-CCR5�/� mice (Fig. 7B). In addition, there was a trendto higher IFNg levels in splenocytes from CpG-treatedMMTV-neu than MMTV-neu-CCR5�/� mice after restimula-tion with the neu (420–429) peptide (P ¼ 0.07; Fig. 7C). CpGalso boosted infiltration of neu-specific (Fig. 7D) and poly-clonal CD8þ T cells (Fig. 7E) into tumor parenchyma; again,this increase was greater in CpG-injected MMTV-neu than intreated MMTV-neu-CCR5�/� mice. FoxP3þ Treg cells wereundetectable in tumors of WT or MMTV-neu-CCR5�/�mice,and their numbers were comparable in dLN from these mice(Supplementary Fig. S10).

Discussion

Reactivation of antitumor adaptive immune responsescould be of clinical benefit, alone or combined with con-ventional cancer therapies (1). We used a number of trans-plantable and primary tumor models to provide geneticevidence that (i) CCR5 agonists, such as CCL5, in the tumorenvironment can inhibit tumor growth in immunocompe-tent hosts, (ii) this effect seems to be CCR5 specific, (iii)CCR5 is a central element in maximizing T cell-mediatedantitumor responses after adoptive transfer of TAA-specificT cells or after immunostimulation with a TLR9 agonist in

tolerized mice, and (iv) the maximal tumoricidal T cell-mediated response requires CCR5 expression in both CD4þ

and CD8þ T cells.Like other inflammatory mediators, the chemokines and

their receptors can either promote or restrict the onset and/orprogression of established tumors (4); CCR5 and its ligandsepitomize this paradox. We analyzed the pro- and antitumoreffects of CCR5 by comparing the growth of subcutaneousgrafts and the incidence of carcinogen-induced and sponta-neous tumors in WT and CCR5-deficient mice. Graft experi-ments with LLC, Panc02, and EG7 tumor cell lines indicatedthat CCR5 expression in host cells restricts progression ofthese subcutaneous tumors. Using the LLC cell line, we showthat the CCR5 antitumor effects are dependent on CCL5 levelsat the tumor site and on the adaptive immune system. Ourresults thus concur with other reports showing that CCR5activation is not a positive determinant for tumor progression(42).

One striking observation was that inhibition of tumor graftsby forced CCL5 expression was CCR5 dependent but CCR1independent, although CCR1 and CCR5 are both expressed oneffector and helper T cells. Differential in vivo roles for CCR1and CCR5 were also described in models for atherosclerosis(43) and renal fibrosis (44), among others. Functional speci-ficity of CCR1 and CCR5 receptors were reported for CCL3-mediated potentiation of the immune response after radio-frequency ablation of murine hepatoma (22); in this case,however, CCL3 antitumor activity was CCR1 dependent butCCR5 independent. These results highlight the functionalspecificity of CCR1/CCR5, which might be linked to tissuecontext or ligand abundance. Moreover, CCR1 and CCR5 havedistinct roles in T-cell transmigration, probably associated tothe differential recycling of these receptors (45). Indepen-dently of the specific mechanism involved, our data reinforcethe concept that the functional redundancy observed in vitroin the chemokine system is even more complex in a pathologiccontext.

In support of a CCR5 antitumor effect, we found enhancedincidence and accelerated onset of MCA-induced sarcomas inCCR5�/� compared with WT mice. CCR5 showed neither aprotective nor a detrimental effect in the onset of spontaneousbreast cancers in MMTV-neu mice. These differences can beexplained by distinct tumor immunogenicity in each model;whereas mice show potent innate and adaptive immuneresponses to MCA-induced tumors (36), MMTV-neu miceare functionally tolerant to neu antigens (40). Forced CCL5expression in fibrosarcoma cells inhibits tumor growth in aCD8þ T cell-dependent manner (42), and we found that MCA-induced sarcoma grew more rapidly in CCR5�/� than in WTmice.

A major conclusion of our study is that, by modulatinghelper-dependent CD8þ T-cell responses, CCR5 is an impor-tant factor in optimizing antitumor immune responses.Effective CD8þ T-cell responses are achieved throughCD4þ T-cell help (33, 34). Although there is a consensusthat CD4þ/CD8þ cooperation requires interaction of bothcell types with the same APC, the temporal regulation ofthese interactions is debated. One model suggests that CD4þ

Figure 7. CCR5 is required for TLR9-mediated reactivation of antitumorresponses. A, growth of untreated and CpG-treated N202.1A tumors inMMTV-neu and MMTV-neu-CCR5�/� mice (n ¼ 6 mice/group). Arrowindicates CpG injection. One representative experiment is shown of 2conducted experiments (*, P < 0.03). B, number of rat-neu (420–429)þ-CD8þ T cells per 2 � 106 cells in dLN of control or CpG-injected mice (*, P¼ 0.04). C, IFNg levels secreted by splenocytes from control or CpG-treated mice after ex vivo restimulation with rat-neu (420–429) peptide.D–E, infiltration of rat-neu (420–429)þ-CD8þ-specific (D) and polyclonalCD8þ T cells (E) into tumors, expressed as the ratio of CpG-treated:untreated mice. B–E, data show mean � SEM from 1 representativeexperiment of 2 (n ¼ 6/group).






and CD8þ cells interact sequentially with the APC (34),whereas in a second model, the CD4þ and the CD8þ cellswould interact simultaneously with a single mature APC thatpresents MHC I- and MHC II peptides (46). On the basis of aperitoneal ovarian cancer model, it was suggested that CCL5produced by CD4þ cells at the tumor site steers CCR5þ DCfor in situ CD40L-mediated licensing (7); although CCR5 wasnot formally implicated in this process, the results of thisstudy support a role for CCR5 agonists in sequential CD4/CD8 cooperation. In contrast, an intravital 2-photon studyshowed that CCL3 and CCL4, produced by DC-CD4þ T cellconjugates at the dLN, guide preactivated naive CD8þ cellsto these complexes and promote simultaneous interaction ofthe 3 cells (6).

Our data for CCR5-deficient OT-I and OT-II cells supportthe second model. Activated OT-I cells preceded OT-II cellsto the tumor site; in our model, DC licensing and cross-priming thus seem not to occur at the tumor site. We alsoobserved a significant reduction in the number of OVA-specific CD8þ cells at the dLN of immunized mice receivingOT-IKOþOT-IIKO cells compared with OT-IWTþOT-IIWT cellrecipients; reduced recruitment to the dLN correlated withthe lower capacity of transferred OT-IKOþOT-IIKO cells torestrict tumor growth compared with CCR5-expressing mix-tures. The finding that CCR5 agonist levels are higher in OT-IIWT-DC than in OT-IIKO-DC complexes suggests a positivefeedback effect, which reinforces the guidance function ofCCR5 when expressed in both cell types. Nevertheless, lackof CCR5 expression in either OT-I or OT-II cells did notaffect recruitment to the dLN, although it impaired OT-I–mediated rejection of EG7 tumors. One interpretation ofthese data is that CCR5 is not only a cell guidance system forCD8þ T cells, which can be partially replaced by otherchemokines/receptor pairs (35), but is also directly involvedin activating the APC, CD4þ, and/or CD8þ cells implicatedin clustering.

In support of this hypothesis, we identify the requirementfor CCR5 expression on CD4þ cells to achieve maximalCD40L upregulation after Ag engagement. In addition,CCR5-expressing CD4þ cells induced more complete DCmaturation than CCR5-deficient counterparts, resulting inenhanced crosspresentation and activation of CD8þ cells.Our results thus indicate an in vivo function of CCR5 inmodulating helper-dependent CD8þ T-cell responses. CCR5not only steers CD8þ T cells (6) but also delivers an earlyactivation signal in CD4þ cells that enhances CD40L/CD40-mediated APC maturation and CD8þ T-cell activation. Thismodel would explain the reduced effectiveness of trans-ferred OT-IWTþOT-IIKO cells in EG7 tumor rejection,although OT-I cell trafficking to dLN after immunizationis unaffected. The observation that CCR5 signaling in T cellsinduces transactivation of NFAT (10), a major transcrip-tional regulator of CD40L (47), argues in favor of direct CCR5regulation of CD40L levels in CD4þ T cells.

The most notable implication of these results is that lackof CCR5 can influence the efficiency of immune-basedcancer therapies, as seen in the reduced response ofMMTV-neu-CCR5�/� mice to the TLR9 agonist. Defective

CCR5 expression in ccr5D32 individuals also reduces cell-mediated immunity to pathogens such as HIV-1 and WestNile virus (11, 12). In the latter case, CCR5 deficiency is nota risk factor for infection, but negatively affects diseaseoutcome (12). Although susceptibility to tolerogeniccancers is independent of CCR5 expression, the progres-sion of some tumors has been associated to CCR5 levels(27, 28).

The CCR5 effect on induction of antitumor immuneresponses might depend on the inflammatory environment.CCR5 expression is reported to counteract the antitumorresponse elicited by a combination of a TLR3 agonist andchemotherapy (48). This differential role of CCR5 in TLR9-and TLR3-mediated immunostimulation suggests integra-tion of local and systemic signaling pathways that affectCCR5 and the TLR. The mechanism underlying this speci-ficity is unknown; some reports pinpoint differences inTLR9- and TLR3-mediated signaling to explain the specificcounter regulation of TLR9-mediated inflammatoryresponses by the glucocorticoid receptor (49) and the differ-ential effects of TLR9 and TLR3 agonists on age-associatedantitumor responses (41) or on arachidonic acid mobiliza-tion (50). TLR9 and TLR3 are thus not wholly equivalent inreactivating the immune system and suggest future studiesto understand the combinatorial control of CCR5 responsesby TLR.

In summary, our study shows that CCR5 is necessary foroptimal activation of adaptive immune responses to tumors.This function might be relevant for cancer therapy and explainthe decreased survival of stage IV melanoma ccr5D32 patientsreceiving immunotherapy compared with patients withoutthe polymorphism (29). Moreover, because the adaptiveimmune system is implicated in the success of some radioand chemotherapy protocols (22, 23), appropriate activationof CCR5, rather than its inhibition, might have broaderapplications for cancer therapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank C. Martínez-A. for critical reading of the manuscript, J. Hern�andezand C. Ardavín for helpful advice and reagents, V. Bronte and R. Brekken forN2021.A and Panc02 cells, respectively, A. Bernad for CCL5 cDNA, F. Ortego forstatistical analysis, and C. Mark for editorial assistance.

Grant Support

This work was supported in part by the Spanish Ministry of Science andInnovation (grant SAF2008-00706), the Carlos III Health Institute RIER Network(RD08/0075), and the Comunidad de Madrid grant IMMUNOTHERCAN (to S.Mañes). A. Gonz�alez-Martín was partially supported by a predoctoral FPIfellowship from the Comunidad de Madrid.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 17, 2011; revised June 24, 2011; accepted June 24, 2011;published OnlineFirst June 29, 2011.






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2011;71:5455-5466. Published OnlineFirst June 29, 2011.Cancer Res Alicia González-Martín, Lucio Gómez, Joseph Lustgarten, et al.

T Cells+ and CD8+Expression in Both CD4Mediated Antitumor Responses Rely upon CCR5−Maximal T Cell

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