CCL5-Mediated Th2 Immune Polarization Promotes …cancerres.aacrjournals.org/content/canres/75/20/4312.full.pdfMicroenvironment and Immunology CCL5-Mediated Th2 Immune Polarization

Microenvironment and Immunology

CCL5-Mediated Th2 Immune PolarizationPromotes Metastasis in Luminal Breast CancerQianfei Zhang1, Jilong Qin2, Lin Zhong3, Lei Gong3, Bing Zhang4, Yan Zhang1, andWei-Qiang Gao1,5

Abstract

The tumor-promoting chemokineCCL5has been implicated inmalignant transformation of breast epithelial cells, with studies todate focusing mainly on basal-type breast cancers. In thisstudy, we investigated the consequences of CCL5 deletion in theMMTV-PyMT transgenic mouse model of luminal breast cancer.In this model, primary tumor burden and pulmonary metastaseswere reduced significantly in CCL5-deficient subjects, an effectfound to be associated with a deficit of Th2 (IL4þCD4þ T) cells.Mechanistic investigations revealed that CCL5 activates CCR3, a

highly expressed chemokine receptor on CD4þ T cells, and alsoboosts Gfi1 expression to promote the differentiation of Th2 cells,which enhance the prometastatic activity of tumor-associatedmyeloid cells. Clinically, polarization toward this immunosup-pressive Th2phenotypewas also evident in patientswith advancedluminal breast cancer. Thus, our findings showed that CCL5/CCR3signaling promotes metastasis by inducing Th2 polarization ofCD4þ T cells,with implications for prognosis and immunotherapyof luminal breast cancer. Cancer Res; 75(20); 4312–21. �2015 AACR.

IntroductionLuminal breast cancer is typically estrogen receptors (ER)þ and

represents the largest proportion of women with breast cancer(around 70%; refs. 1, 2). Although luminal breast cancer patientsrespond to endocrine therapy, those with high ERa levels inev-itably develop early and late relapses andmetastasis. Discovery ofnew therapeutic targets for the treatment of these patients repre-sents a big unmet medical need (1, 2).

CCL5/RANTES is one of the CC chemokine family proteins andinteracts with G-protein–coupled receptors CCR1, CCR3, andCCR5 in various types of cells (3). Several studies have exploredthe association of CCL5 levels with breast cancer progression, anddemonstrated that CCL5 expression is positively correlated withthe disease stages at stages II and III (4, 5, 6). Our previous dataalso showed that host-derived CCL5 promotes breast cancergrowth and metastasis by restraining the normal differentiationof MDSC subsets in a 4T1 mammary carcinoma model thatmimics the triple-negative breast cancer (TNBC) in the clinic(7). However, whether and how CCL5 promotes progression andmetastasis of luminal breast cancer has not been determined.

In this study, we investigated that roles of CCL5 in luminalbreast cancer and found that CCL5 exerted its effects via CCR3 onCD4þ T cells and increased Gfi1 expression to induce the Th2polarization of CD4þ T cells, which in turn promoted pulmonarymetastasis of luminal breast cancer. In addition, there was apositive correlation between not only the tumor tissue expressionlevels of CCL5 (or IL4) or the serum CCL5 protein level (orperipheral blood Th2 cell numbers) and the progressiveness ofclinical luminal breast cancer patients. These findings indicatethat not only CCL5 can be a therapeutic target for the treatment ofluminal breast cancer, but also serum CCL5 level and/or Th2 cellnumbers in the peripheral blood might be new noninvasiveprognostic indicators for monitoring the progression of luminalbreast cancer.

Materials and MethodsPatients

Primary breast carcinomas were obtained from patients at thefirst affiliate hospital of Guangzhou Medical University (Guangz-hou, China). Blood samples of 11 luminal breast cancer patients,5 TNBC patients and 5 healthy human donors were gotten fromRenji Hospital (Shanghai, China) and Yuhuangding Hospital(Yantai, China).

MiceMMTV-PyMT in background of FVB/n and CCL5�/� mice in

background of BALB/c were obtained from The Jackson Labora-tory. To generate CCL5�/� PyMT mice, CCL5�/� mice were back-crossed into the FVB/n strain to N8, and then intercrossed withPyMT mice.

Study approvalAll animal studies were reviewed and approved by the Insti-

tutional Animal Care and Use Committee of Shanghai JiaotongUniversity. All human samples were collected with the informedconsent of the patients and the procedureswere approvedbyRenjiHospital of Shanghai Jiaotong University (Renji [2013] N013).

1State Key Laboratory of Oncogenes and Related Genes, Renji-Med XClinical Stem Cell Research Center, Ren Ji Hospital, School of Bio-medical Engineering, Shanghai Jiao Tong University, Shanghai, China.2Department of Pathology, The First Affiliate Hospital of GuangzhouMedical University, Guangzhou, China. 3Yuhuangding Hospital,Yantai,China. 4Department of Cardiology, Boston Children's Hospital, Bos-ton, Massachusetts. 5Collarative Innovation Center of SystemsBiomedicine, Sanghai, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Q. Zhang and J. Qin contributed equally to this article.

Corresponding Authors:Wei-Qiang Gao or Yan Zhang, Ren Ji Hospital Stem CellResearchCenter, Shanghai JiaoTongUniversity, 160PujianRoad, ShanghaiChina,200127. Phone: 86-158-2112-6637; Fax: 86-21-6838-3916; E-mail:[email protected]; and Yan Zhang, [email protected]

doi: 10.1158/0008-5472.CAN-14-3590

�2015 American Association for Cancer Research.

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Immune cell isolationLymph nodes and spleens were mechanically dissociated and

strained through a 40-mm nylon mesh to produce a single cellsuspension. After RBCs were lysed, CD4þ T cells were isolatedwith the CD4þ T-cell Isolation Kit II (Miltenyi Biotec). ForMDSCs, cells were stained with CD11b and Gr-1 antibodies, andthen sorted by flow cytometry. For na€�ve CD4þ T cell, cells wereisolated with the CD4þCD62Lþ T-cell Isolation Kit II (MiltenyiBiotec). For tumor-infiltrating leukocytes (TIL), tumors weredigested into single cells with collagenase type II (0.5 mg/mL),collagenase type IV (0.5 mg/mL), hyaluronidase (10 U/mL), andDNase I (0.01 mg/mL) for 2 hours at 37�C. The dissociated cellswere collected, lysed by RBC lysis buffer, and then incubated withCD45 monoclonal antibody. For tumor-associated macrophages(TAM), the cells were incubated with F4/80 and CD11b mono-clonal antibody. The positive cells were next sorted by flowcytometry on BD FACSAria using BD FACSDiva software. Thepurity of the isolated subpopulations regularly exceeded 90%.

Cell culture conditionA total of 1.5� 106 na€�ve CD4þ T cells were activatedwith 1.0�

107 T-depleted splenocytes as APC with 3 mg/mL anti-CD3 and 3mg/mL anti-CD28 Abs. For Th2 differentiation the cells werecultured with 10 ng/mL IL4 and 10 mg/mL anti-IFNg . For serum-induced differentiation of na€�ve CD4þ T cells, IL4 and anti-IFNgwere replacedby50%V/V serum fromMMTV-PyMTmice. The cellsweredifferentiated for 1weekand the culturemediumwas changedevery 2 days. For the study of signaling pathway, na€�ve CD4þ T cellsfrom spleens of CCL5þ/þ and CCL5�/� mice were stimulated withT-depleted splenocytes under Th2 condition for 3 days and culturewith only IL2 for 1 day. Then cells were reactivated by T-cell–depleted splenocytes under Th2 condition for 1 hour (Erk/MAPKand Stat6) or for 4 hours (Gfi1 and Gata3). For intracellularcytokine staining, T cells were restimulated with leukocyte activa-tion cocktail and protein transport inhibitor (BD Biosciences), andstained according to the manufacturer's directions (eBioscience).

3D organotypic cocultureMouse mammary epithelial cells (MEC) were isolated as pre-

viously described (8). Briefly, the fourth mammary fat pads from12-week-old CCL5þ/þ PyMT or CCL5þ/þ PyMT were harvested.Then tissues were minced finely and digested with Collagenase A3.0 mg/mL (Roche) and Trypsin 1.5 mg/mL (Gibco). Three-dimensional (3D) organotypic cultures were established as pre-viously described (9, 10), and medium containing 10% Matrigelwas replaced every 4 days. Coculture with TAM or CD4þ T wasestablished when stable organoid formed about 2 weeks later.Immune cells were overlaid in medium containing 2% Matrigeland the percentage of invasion acini was assessed 2 days later.

Statistical analysisThe Student t test was used to analyze the data. Results are given

as mean � SEM unless otherwise indicated. P values <0.05 wereconsidered significant.

ResultsCCL5 expression is positively correlated with disease progressionin an MMTV-PyMT mouse model of luminal breast cancer

We used an MMTV-PyMT transgenic mouse line, a reliablemodel of human luminal breast cancer (11), to study the roleof CCL5 in this major type of breast cancer. By 5 weeks, female

carriers developed palpable mammary tumors that were multi-focal and involved the entire mammary fat pad. Approximately94% of tumor-bearing females developed pulmonary metastasisby 3 months of age (12, 13). CCL5 expression in the tumor sitewas examined by IHC at ages of 5, 11, and 18 weeks, respectively.As expected, CCL5 expression was increased progressively duringbreast cancer tumorigenesis in the MMTV-PyMT mice (Fig. 1A),similar to the pattern of human breast cancer progression in theclinic (14).

Deletion of CCL5 inhibits luminal breast tumorigenesis andpulmonary metastasis in the MMTV-PyMT mouse model

To determine the significance of CCL5 expression in breastcancer, we generated PyMTmice bearing homozygous null CCL5(CCL5�/� PyMT). Notably, primary mammary tumor latencywas dramatically longer in CCL5�/� PyMT compared with thecontrol cohort CCL5þ/þ PyMTmice (Fig. 1B). The tumor burdenat an age of 11 weeks was significantly decreased due to the lossof CCL5 expression whereas at an age of 18 weeks such differ-ence almost disappeared (Fig. 1C). However, the pulmonarymetastasis of 18-week CCL5�/� PyMT mice was dramaticallyattenuated in both the tumor burden in the lung and thenumber of metastatic foci (Fig. 1D–F). To further confirm theattenuated metastasis in CCL5�/� PyMT, we also examinedthe circulating tumor cell numbers (CTC, cytokeratinþ/CD45�)in the peripheral blood and found that the number of CTC wasalso decreased significantly (Fig. 1G). These data indicate thatdeletion of CCL5 inhibits not only luminal breast tumorigenesisbut also the pulmonary metastasis.

Th2 cells mediate a metastasis advantage of CCL5 in luminalbreast cancer

Because the reduced pulmonary metastatic phenotype in theCCL5�/� PyMT is similar to that of PyMT/CD4�/�mice reportedpreviously by the Lisa M. Coussens's group (9), we determinedwhether CCL5 deficiency would affect the polarization of CD4þ

T cells in the progression of mammary carcinoma. TILs werefirst evaluated by flow cytometry and IHC analysis, for CCL5 iswell known as a chemokine to recruit leukocytes to the site ofinfection. As shown in Fig. 2A–C, there was no differencebetween the numbers and the composition of CD45þ cellsinfiltrating primary tumors of 18-week old CCL5þ/þ PyMT andCCL5�/� PyMT mice.

Then we isolated CD4þ T cells from TILs and tumor-draininglymph nodes (LN) of 18-week old CCL5þ/þ PyMT and CCL5�/�

PyMTmicebymagneticmicrobeads, andexamined the expressionof specific transcriptional factors by RT-PCR. At the same time,these isolated CD4þ T cells were ex vivo activated by leukocyteactivation cocktail for 18 hours, culture medium were collectedand effector cytokines indicative of Th1, Th2, Th17, and Tregresponse were examined by ELISA. As shown in Fig. 2D, GATA3(Th2) expressionwas dramatically decreased inCD4þ T cells fromboth TILs and LNs of CCL5�/� PyMT mice, whereas other tran-scriptional factors examined had no significant changes betweenthese two cohorts except for the elevated expression of T-bet (Th1)in LNs. These results were echoed by the expression of effectormolecules of a different subtype CD4þ T cells (Fig. 2E), indicatingthat tumor-induced Th2 polarization of CD4þ T cells was inhib-ited in CCL5�/� PyMT mice. To further confirm these results,CD4þ T cells from tumor-draining LNs were ex vivo activated byleukocyte activation cocktail for 4 hours. Then effector cytokine

CCL5 Promotes Th2 Polarization in Luminal Breast Cancer

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expression was examined by intracellular flow cytometry andthese analyses revealed that the percentage of IL4þCD4þ T cells(Th2) was markedly decreased (Fig. 2F and G). In conclusion,CCL5 deficiency in MMTV-PyMT mice inhibited CD4þ T cellspolarization to Th2 cells during the progression of mammarycarcinoma.

To explore the role of CCL5-induced Th2 polarization ontumor latency and primary tumor burden, we checked IL4 expres-sion of CD4þ T cells isolated from spleen, tumor-associated LN(or axillary LN), TIL and lung at age of 6, 11, and 18 weeks.Because the great differences on tumor latency, tumorburden, andpulmonary metastasis between CCL5þ/þ PyMT and CCL5�/�

PyMTmice, respectively, existed at those time points, and we alsoexamined the number of CTC cells in peripheral blood tomonitorthe status of tumor metastasis at each time point. The results ofqPCR showed that only at age of 18 weeks, the mRNA level of IL4was strongly increased in all of the CD4þ T cells ofCCL5þ/þ PyMTmice, compared with CCL5�/� PyMT mice (SupplementaryFig. S1), suggesting that lower level of Th2 cells may contributeto the decreased pulmonary metastasis, but not the increasedtumor latency and reduced primary tumor burden of CCL5�/�

PyMT mice.To further confirm that the altered polarization of CD4þ T cells

contributes to the reduced lungmetastasis inCCL5�/�PyMTmice,we depleted T-cell subsets in vivo during carcinogenesis ofCCL5þ/þ PyMT and CCL5�/� PyMT mice by i.p. injection of250 mg anti-CD4 antibody (GK1.5) on days 85, 86, 89, and 92,respectively (the efficiencyofCD4þT cells depletionwas shown inSupplementary Fig. S3A). As shown in Fig. 2H and I, blocking ofCD4þ T cells resulted in a significantly reduced number ofpulmonary metastatic foci in CCL5þ/þ PyMT mice compare withthe controls, and consequently the difference of pulmonarymetastasis induced by CCL5 deficiency disappeared. The findingsshowing that Th2 cells are themajor subtype ofCD4þT cells in the

tumor microenvironment of CCL5þ/þ PyMTmice argue against apossible involvement of other effector lineage of CD4þ T cells(Fig. 2F and G). To provide further supporting evidence for thisinterpretation, we examined the number of CTCs by flow cyto-metry. Consistent with the pulmonary metastasis analysis, therewas no significant statistical difference in the number of CTCsbetween CCL5þ/þ PyMT and CCL5�/� PyMTmice after depletionof CD4þ T cells (Fig. 3J). Collectively, these data suggested thatTh2 cells play a major role in CCL5-induced pulmonary metas-tasis of luminal breast cancer.

Tumor-associated myeloid cell phenotype induced by CCL5 isCD4þ T cell dependent

Previous studies reported that tumor-associated myeloid cells,including TAMs and MDSCs, promote tumor cell invasion, andmetastasis, which can be modulated by communicating with Th2cells (15, 16). Todeterminewhether thesemyeloid cell phenotypeand effector bioactivity are affected by CCL5 deficiency and whatis the relationship with CD4þ cells, we isolated TAMs fromprimary mammary tumor or MDSCs from spleens of bothCCL5þ/þ PyMT and CCL5�/� PyMT mice and compared theirexpression levels of several cytokines indicative of macrophagespolarization or epithelial cell invasion. As shown in Supplemen-tary Fig. S2A, the levels of cytokines, TGFb and EGF, were signif-icantly lower in the TAMsof theCCL5�/�PyMTmice, as comparedwith that of the control mice. The same tendency was observed inMDSCs (Supplementary Fig. S2B and S2C). However, followingthe blocking of the CD4þ T cells by using CD4-neutralizingantibody, the distinctive expression of cytokines and growthfactors in TAMs and MDSCs between CCL5þ/þ PyMT andCCL5�/� PyMTmice became largely diminished (SupplementaryFig. S3B and S3C), together indicating that it is CD4þ T cells thatmediate the effect of CCL5 on activities of tumor-associatedmyeloid cells in MMTV-PyMT mice.

Figure 1.CCL5 deficiency inhibits luminal breastcancer tumorigenesis and metastasis.A, histologic identification of murineCCL5 staining in the tumor of MMTV-PyMTmice. B, Kaplan–Meier tumor-freesurvival curves of MMTV-PyMT mice. C,total tumor burden ofMMTV-PyMTmice(n¼ 10) wasmeasured at 11 or 18weeks.D, quantitation of lung metastasisdetermined by counting of foci per lunglobe (n¼ 10). E, histologic identificationof tumor burden. F, representativepictures of lungs from MMTV-PyMTmice. G, CTCs were analyzed by flowcytometry. ��, P < 0.01; �� , P < 0.005.

Zhang et al.

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CD4þ T cells from CCL5�/� PyMT mice inhibit invasion ofmammary carcinoma cells in 3D organoid cultures byregulating properties of tumor-associated myeloid cells

To explore themechanism on how CD4þ T cells from CCL5�/�

PyMT mice inhibits tumor invasion, we first established an exvivo 3D organotypic culture model with primary MECs derivedfrom PyMT mice. As described previously (10), MECs werecollected from 12-week-old CCL5þ/þ PyMT or CCL5�/� PyMTmice, placed in growth factor–reduced Matrigel and allowed toform stable organoids for 2 weeks and the effects of CD4þ Tcells and tumor-associated myeloid cells from CCL5�/� PyMTmice on MECs' invasion were first examined in this 3D cocul-ture system. To simplify the experiment, we only chose TAMs as

a potential mediator of CD4þ T lymphocytes. After 3 days,TAMs from CCL5þ/þ PyMT dramatically enhanced organoiddisruption of either CCL5þ/þ or CCL5�/� MEC, whereas TAMsfrom CCL5�/� PyMT had no significant effects on the invasivephenotype of MECs (Fig. 3A). Morphologically, the organoidscocultured with CCL5þ/þ TAMs had more spindle-shaped cells(Fig. 3B). These data suggest that TAM from CCL5�/� PyMTmice lost the proinvasive activity, whereas loss of CCL5 inMECs did not affect their invasive activity, compared with thecontrol group.

To further confirm the regulatory function of CD4þ T cells onmetastatic activity of tumor-associated myeloid cells, these cellsisolated from tumor-draining LNs were cocultured with MEC

Figure 2.CD4þ T cells confer the reduced metastasis in CCL5-null mice. A, analysis of the CD45þ cells percentage in tumors by flow cytometry. B, histologic identificationof CD45þ staining in the tumors. C, flow cytometric analysis of leukocyte population of TILs; (n ¼ 6). D, relative mRNA level of T-bet, GATA3, RORgt, andFoxP3 of CD4þ T cells isolated from TIL and LNs. E, protein level of IFNg , IL4, IL17, and IL10 in the culture medium of CD4þ T cells. F, FACS analysis of cytokinessecreted by CD4þ T cells ex vivo. G, quantification of F. H, quantitation of lung metastasis determined by counting of foci per lung lobe from 100-day-oldMMTV-PyMT mice (n ¼ 5) following i.p. injection of anti-CD4. I, histologic identification of tumor burden in the lungs. J, CTCs were analyzed by flow cytometry.� , P < 0.05; �� , P < 0.01; ns, nonsignificant.






organoids in the presence of CCL5þ/þ or CCL5�/� TAMs. Asexpected, the percentage of the invasive organoids induced byeither CCL5þ/þ or CCL5�/� TAMs was significantly increased inthe presence of CCL5þ/þ CD4þ T cells and exogenous CCL5protein could not mimic the function of CD4þ T cells (Fig. 3Cand D). On the contrary, CD4þ T cells from CCL5�/� PyMTinhibited proinvasive activity of MECs mediated by CCL5þ/þ

TAMs, indicating that a prometastatic activity of TAMs is furtherregulated by the status of CD4þ T cells (Fig. 3C and D). Moreimportantly, in the absence of TAMs, CD4þ T cells from eitherCCL5þ/þ PyMT or CCL5�/� PyMT alone had no effect on theinvasion of MEC organoids (Fig. 3C and D, close bar). These datasuggest that CD4þ T cells from CCL5-null mice inhibit MECorganoid invasion through modulating activity of tumor-associ-ated myeloid cells.

To further explore the mechanism on the lower invasiveactivity of MECs induced by CCL5�/� CD4þ T cells, we exam-ined the expression of EMT (epithelial–mesenchymal transi-tion)-related genes in MEC organoids, for EMT allows thepolarized epithelial cancer cells to acquire the mesenchymalcell phenotype, such as enhanced migratory capacity and inva-siveness (17). The results showed that the expression ofE-cadherin was higher and the expressions of vimentin andZeb1 were lower in MEC organoids that cocultured withCCL5�/� CD4þ T cells, compared with those with control cells(Fig. 3E; Supplementary Fig. S4). These results were furtherconfirmed by qPCR data of cancer cells from 18-week-old mice(Supplementary Fig. S5), suggesting that the lower rate ofconversion from epithelia to mesenchyme may contribute toantiinvasive activity of CCL5�/� CD4þ T cells.

CCL5 promotes Th2 cell differentiation in vitroTo determine whether loss of Th2 cells in CCL5�/� PyMTmice

was attributable to the reduced differentiation of Th2 cell in theabsence of CCL5 protein, we examined Th2 polarization of CD4þ

T cells under CCL5-null conditions in vitro. Western blottingrevealed that lots of CCL5 protein is expressed in CD4þ T cells(Supplementary Fig. S6), suggesting that the autocrined CCL5protein from T cells and feeder cells is sufficient for inducing Th2

polarization in vitro. Na€�ve CCL5�/� CD4þ T cells or wild-typecontrols were, therefore, activated under Th2 or Th0 conditionsfor 7 days, and cytokine phenotypes were examined. As shownin Fig. 4A and B, CCL5 deficiency dramatically inhibited thegeneration of IL4þCD4þ T cells under Th2 conditions, whereasCCL5 protein alone could not induce na€�ve T cells to Th2 differ-entiation under Th0 conditions. To further test whether exoge-nous CCL5 can rescue the defect on Th2 cells polarization ofCCL5-null CD4þ T cells, recombinant murine CCL5 proteinwas added back and the addition of CCL5 at a concentration of20 ng/mL almost completely recovered the Th2 polarization ofCCL5-null CD4þ T cells (Fig. 4A and B). Collectively, eitherendogenous or exogenous CCL5 protein could cooperate withboth IL4- and IFNg-neutralizing antibody to facilitate the gener-ation of Th2 cells.

To further explore the role of CCL5 on CD4þ T-cell differ-entiation in vivo, we induced the differentiation of na€�ve CD4þ

T cell with the serum from 18-week-old CCL5þ/þ PyMT orCCL5�/� PyMT mice, instead of IL4 and anti-IFNg . Flow-cytometry data showed that the percentage of Th2 cells wasdramatically decreased in CCL5�/� CD4þ T cells, but those ofother subsets were not significantly increased, although per-centages of Th1 were slightly increased, compared with thecontrol group (Fig. 4C and D). These results suggested thatCCL5 mainly promotes the Th2 differentiation, but not othersubsets of CD4þ T cells, in the mouse model of luminal breastcancer.

Th2 differentiation is promoted by CCL5-induced Gfi1expression

To figure out the mechanism underlying CCL5-induced Th2differentiation of CD4þ T cells, we suspected growth factorindependent-1 (Gfi1), a zinc-finger transcriptional repressor(18), as a possible molecule involved in this process, becausethe phenotype of Gfi1-knockout mice is similar to that of CCL5null mice bearing 4T1mammary carcinoma, that is, generation ofatypical CD11bþGr-1þ cells is induced by cytokines secreted bytumor cells (7, 19, 20). In addition, Gfi1 has been proven to playan essential role in CD4þ T cells polarization (21). To explore the

Figure 3.CCL5�/�CD4þ T cells inhibit invasion ofmammary carcinoma cells in 3Dorganoid cell culture by modulatingproperties of TAM. A, quantitation ofthe percentage of invasive organoidsfollowing MEC coculture with TAM. B,representative bright field images ofMEC spheres in the cocultures withTAM. C and D, quantitation of thepercentages of invasive organoidsfollowing MEC coculture with CCL5þ/þ

TAM (C) or CCL�/� TAM (D) and CD4þ

T cells. E, relative mRNA level of E-cadherin, vimentin, and Zeb1 from MECorganoids. � , P < 0.05; ��, P < 0.01.

Zhang et al.





role of Gfi1 in CCL5-induced Th2 polarization, we first investi-gated the endogenous Gfi1 expression in CD4þ T cells. Theseassays showed that the endogenous Gfi1 and IL4 expression inCD4þ T cells isolated from the tumor-associated leukocyteswere dramatically decreased in CCL5-null tumor-bearing mice(Fig. 5A).

Because Gfi1 can be induced by IL4 during Th2 polarization ofCD4þ T cells (21), its induction in CCL5�/� CD4þ T cells wasstudied in detail. As shown in Fig. 5B, although there was notmuch difference between these two groups by 3days of induction,the amount of Gfi1 protein in CCL5�/� cells was significantlylower than that ofCCL5þ/þ cells on day 7 (Fig. 5B; SupplementaryFig. S7A). To further confirm these results, rmCCL5 protein wasadded back to the cell culturemediumofCCL5�/�CD4þ T cells atincreased doses and the expression of Gfi1 protein was graduallyincreased to the comparable level of that inCCL5þ/þ cells (Fig. 5C;Supplementary Fig. S7B). These results suggest that CCL5 mightact as an upstream ligand to induce Gfi1 expression to promoteTh2 polarization of CD4þ T cells.

To determine whether reintroduction of Gfi1 rescues thedefect on Th2 polarization in CCL5�/� cells, we infected iso-lated na€�ve CCL5�/� CD4þ T cells with lentiviruses encodingGfi1 and GFP and induced these cells to differentiate into Th2cells. The efficiency of lentiviral infection was determined byWestern blotting of the Gfi1 protein and fluorescence micros-copy of GFP expression (Supplementary Fig. S8). After 7 dayspost-lentiviral infection, the cells infected with Gfi1-expressing

lentivirus had higher IL4 expression than those with the controlGFP lentivirus (Fig. 5D). Therefore, the defect in Th2 polari-zation, resulting from loss of endogenous CCL5, could becorrected by reexpression of Gfi1.

To further explore the signaling pathways involved in CCL5-induced Gfi1 expression, we examined the activation of Erkand Stat6 in CCL5þ/þ and CCL5�/� T cells that were reactivatedby T-cell–depleted splenocytes under Th2 condition for 1hour, following 3 days in culture under Th2 condition and1 day in culture with only IL2. Because TCR-mediated activa-tion of Erk- and IL4-mediated activation of Stat6 have beendemonstrated to involve the Gfi1 expression during Th2 polar-ization of CD4þ T cells (21, 22). The results of WesternBlotting showed that the activation of Erk was not affected, butthat of Stat6 was dramatically reduced in CCL5�/� T cells com-paredwith the control group, alongwith the decreased expressionof Gfi1 and Gata3 (Fig. 5E). These data suggested that the IL4–Stat6 signaling pathway, not the TCR–Erk pathway, might beinvolved in theCCL5-inducedGfi1 expression in Th2polarizationof CD4þ T cells.

CCR3 is the functional receptor mediating CCL5-induced Th2differentiation

It is well known that CCL5 induces leukocyte migration viathree high-affinity receptors of the GCPR family, namely CCR1,CCR3, andCCR5 (23). Real-timeRT-PCR revealed that expressionof CCR3 was much higher in CD4þ T cells and TILs than that in

Figure 4.CCL5 enhances generation of Th2 cells in vitro. A and B, FACs analysis of IL4þCD4þ T cells in CD4þ T cells after 7 days of Th2 polarization. Shown in B is thepercentage of IL4-positive cells in A. C and D, IFNg , IL4, FoxP3, and IL17 staining of CD4þ T cells. The protocol is same as A and B, except for replacing Th2-inducingcytokines and antibodies with serums from tumor-bearing mice. � , P < 0.05; �� , P < 0.01.






tumor cells (Supplementary Fig. S9). To further determine wheth-er these three receptors are all involved in the pro-Th2 cellpolarization activity of CCL5, we examined their expressionpattern during Th2 differentiation in CD4þ T cells by RT-PCR.The transcript of CCR1 from na€�ve CCL5þ/þ CD4þ T cells was setas 1 and others were shown in values relative to that of CCR1. Asshown in Fig. 6A–C, all of transcripts for CCR1, CCR3, and CCR5were almost undetectable in freshly isolated na€�ve CD4þ T cells.However, there were somedifferences in the subsequent days: Thelevels of mRNAs for CCR1 were barely detectable at days 3 and 7,whereas these for CCR3 were dramatically increased at day 3 andkept on at high levels until day 7. For CCR5, it became readilymeasurable at days 3 and 7, but the expression level was signif-icantly lower compared with the transcript of CCR3. To furtherconfirm the expression pattern of CCR3 and CCR5 on CD4þ Tcells, we also examined their expression by immunofluorescencestaining (Fig. 6D and E). These results were consistent with theqPCR data as well as the previous report showing that specificchemokine receptors are associated with polarized subsets ofCD4þ T cells and that CCR3 is mainly expressed in Th2 polari-zation (24).

Next, we used inhibitors of chemokine receptors to determinewhich of these receptors is important for CCL5-induced Th2differentiation. Na€�ve CD4þ T cells from WT mice were treatedunder the Th2 condition for 2 days before the addition of a CCR3or CCR5 inhibitor into the cell culture medium. Not surprisingly,

the CCR3 inhibitor (SB328437) significantly suppressed the Th2polarization of CD4þ T cells, whereas the CCR5 inhibitor (Mar-aviroc) had little effect on it (Fig. 6F and G).

Considering the fact that our culture system contained someAPCs that might affect the Th2 polarization via secretion ofIL12 (25), we performed an experiment by adding IL12p40-neutralizing antibody to the Th2-induction medium. As shownin Fig. 6F and G, inactivation of IL12 did not significantlyaltered CCL5-induced Th2 differentiation. These results suggestthat CCL5 promotes Th2 polarization by directly activatingCCR3 on CD4þ T cells, rather than via an indirect contributionfrom APCs.

High expression of CCL5 and IL4 is relevant to theaggressiveness of clinical luminal breast cancer

Previous studies showed that CCL5 expression is positivelycorrelated with human breast cancer progression only at stagesII and III (4, 5). To validate the correlation between increasedexpression levels of CCL5 and IL4 and the tumor grades ofhuman luminal breast cancer samples, we took tissues fromparaffin-embedded clinical samples of both normal and lumi-nal breast tumors (stages II and III) and conducted a TaqManqRT-PCR assay to measure their expression. We found that theexpression of both CCL5 and IL4 were significantly higher inmore aggressive tumors than less malignant tumors or thenormal breast tissue (Fig. 7A and B), and the correlation

Figure 5.Promotion of Th2 differentiation byCCL5depends onGfi1 expression.A, theprotein level of Gfi1 expression in CD4þ

T cells from tumor-infiltratedleukocytes. B and C, Western blottingof Gfi1 expression. Na€�ve CD4þ T cellsfrom spleens of CCL5þ/þ and CCL5�/�

mice were stimulated under Th2condition for indicated time (B). CCL5proteinwas added back into the culturemedium. C and D, rescue of Th2differentiation from CCL5�/� CD4þ Tcells by lentiviral transfer of Gfi1. E,Western blotting of Erk and Stat6signaling pathways.

Zhang et al.





analysis revealed that expression levels of CCL5 and IL4 werepositively correlated (Pearson's r ¼ 0.75; Fig. 7C). Furthermore,we observed a direct correlation between CCL5 and IL4 expres-sions in the breast cancer tissue by immunohistochemistry andboth of them were positively associated with the malignantgrades of breast cancer (Fig. 7D). Clinically, based on Her2expression, ERþ luminal breast cancer can be divided into twotypes: luminal A (Her2�) and luminal B (Her2þ). So wegrouped these breast cancer patients according to their molec-ular markers and examined expression levels of CCL5 and IL4between these two groups (Supplementary Fig. S10A andS10B). The results showed that there was no discriminationbetween luminal A and luminal B types.

Serum CCL5 levels and numbers of peripheral blood Th2 cellsare positively correlated with aggressiveness of human luminalbreast cancer

To provide further evidence that CCL5 enhances Th2 polari-zation, we examine the correlation between CCL5 expression andthe number of Th2 cells in the peripheral blood of luminal breastcancer patients. The results demonstrated that serum CCL5 levelsand percentages of IL4þCD4þ T cells in the peripheral blood weresignificantly higher in luminal breast cancer patients at stage III ascompared with stage II (Fig. 7E and F; Supplementary Fig. S10C).The correlation analysis showed that serum CCL5 levels and the

numbers of peripheral IL4þCD4þ T cells were positively corre-lated in malignant breast cancer patients (Pearson's r ¼0.6229; Fig. 7G).

DiscussionPrevious studies mainly focused on roles of CCL5 in basal

breast cancer and interpreted its role via differentmechanisms. Forexample, CCL5 recruits the accumulation of deleterious TAMs inthe mammary carcinoma site and promotes its expression ofMMP9 to facilitate the disease progression (14). In addition toindirectly potentiating cancer development by regulating prop-erties of immune cells, research from several laboratories hasrevealed that CCL5 regulates tumor growth and invasion throughdirect activation of CCR5 receptors on MECs. For instance, Kar-noub and colleagues (26) demonstrated that MSC-derived CCL5promotes human breast cancer cell invasion and release of MMPsby binding to CCR5 directly on cancer cells in a xenograft breastcancer model. On the other hand, it is important to point out thatmost of these studies have been done in basal breast cancermodels (2). The present study shows clear evidence that CCL5also plays an important role in luminal breast tumorigenesis andmetastasis via a differentmechanism, that is CCL5 exerts its effectsvia induction of Th2polarization, rather than via direct binding tothe myeloid cells or the tumor cells. Therefore, the present work

Figure 6.CCR3 on CD4þ T-cell membrane mediates CCL5-induced Th2 polarization. A–C, na€�ve CD4þ T cells from CCL5þ/þ and CCL5�/� mice were cultured in theTh2 condition for up to 7 days and tested for chemokine receptors CCR1, CCR3, and CCR5 expression by RT-PCR. D and E, immunofluorescence analysis ofCCR3 (D) and CCR5 (E) distribution in na€�ve CD4þ T cells and Th2 cells. DAPI, blue; CCR3, green; CCR5, red. F, na€�ve CD4þ T cells from WT mice werecultured in the Th2 condition; after 2 days the culture medium was added with DMSO or 30 nmol/L CCR3 inhibitor, or 100 nmol/L CCR5 inhibitor or 10 mg/mLanti-IL12p40 antibody. G, quantification of F. �� , P < 0.01.






sheds new light on the role of CCL5 during tumorigenesis andmetastasis of breast cancer, in particular, the luminal breastcarcinomas

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. ZhangDevelopment of methodology: Y. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhong, L. Gong, B. Zhang, Y. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q. Zhang, J. Qin, L. Zhong, L. Gong, B. Zhang,Y. Zhang, W.-Q. Gao

Writing, review, and/or revision of themanuscript:Q. Zhang, J. Qin, Y. Zhang,W.-Q. GaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. ZhangStudy supervision: Y. Zhang, W.-Q. Gao

Grant SupportThis work is supported by funds from the National Natural Science Foun-

dation of China (81201524 and 81372257 to Y. Zhang; 81130038 and81372189 to W.Q. Gao).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 11, 2014; revised June 1, 2015; accepted July 23, 2015;published OnlineFirst August 6, 2015.

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Figure 7.High CCL5 and IL4 expressions are associated with advanced human breast cancer. A and B, expression of CCL5 and IL4 was measured by TaqMan qRT-PCRafter microdissection. C, correlation between CCL5 and IL4 expressions in luminal breast cancer. D, representative images of CCL5 and IL4 staining in breast cancer.E, serum CCL5 was measured by ELISA. F, representative flow cytometric figures of IL4þCD4þ T cells in the peripheral blood. G, correlation betweenserum CCL5 levels and peripheral blood Th2 cell numbers in luminal breast cancer.

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2015;75:4312-4321. Published OnlineFirst August 6, 2015.Cancer Res Qianfei Zhang, Jilong Qin, Lin Zhong, et al. Luminal Breast CancerCCL5-Mediated Th2 Immune Polarization Promotes Metastasis in

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