Molecular Cancer Therapeutics - CCR5-Dependent Homing of ......2017/11/17 · mouse squamous cell carcinoma (SCC) development (5). How-ever, the molecular basis dictating the homing

Cancer Biology and Translational Studies

CCR5-Dependent Homing of T Regulatory Cells tothe Tumor Microenvironment Contributes to SkinSquamous Cell Carcinoma DevelopmentCarine Ervolino de Oliveira1, Thaís Helena Gasparoto2, Claudia Ramos Pinheiro2,N�adia Ghinelli Amor2, Maria Renata Sales Nogueira3, Ramon Kaneno4,Gustavo Pompermaier Garlet2, Vanessa Soares Lara1, Jo~ao Santana Silva5,Karen Ang�elica Cavassani6, and Ana Paula Campanelli2

Abstract

Squamous cell carcinoma (SCC) is one of the most commonhuman cancers worldwide. Recent studies show that regulatoryT cells (Treg) have a critical role in the modulation of anantitumor immune response, and consequently the SCC devel-opment. Because the accumulation of Tregs at the tumor site is,in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in theSCC development. Our findings showed that CCR5-deficientmice (CCR5KO) were efficient in controlling papilloma's inci-dence when compared with wild-type mice. Analysis of tumorlesions in wild-type (WT) and CCR5KO mice revealed that lackof CCR5 lead to significant reduction in frequency of Tregs andincreased of CD4 T cells into the tumors. Moreover, the adop-

tive transfer of naturally occurring Tregs CD4þCD25þCCR5þ,CD4þCD25�CCR5þ or CD8þCCR5þ conventional T cells toCCR5KO mice resulted in an increased papilloma incidence.Interestingly, adoptive transfer of WT CD4þCD25þCCR5þ cellsto CCR5KO mice induced more undifferentiated SCC lesions,characterized by higher infiltration of macrophages and den-dritic cells. In this study, we also demonstrated that Tregmigration to the tumor microenvironment is mediated byCCR5, and these cells are promoting tumor growth via inhi-bition of antitumor cells such as cytotoxic CD8þ T cells. Ourfindings reinforce the therapeutic potential of CCR5 inhibitionfor cancer treatment, and indicate an attractive approach forSCC treatment. Mol Cancer Ther; 16(12); 1–10. �2017 AACR.

IntroductionRegulatory T cells (Treg) play an important role in the main-

tenance of peripheral tolerance and the modulation of immuneresponses against infections and tumor cells. Immunosuppres-sion in the tumor microenvironment mediated by Tregs is animportant mechanism of tumor immune escape (1). Recruitmentof Tregs favors tumor progression and depletion of these cellsenhances antitumor immune responses (2–6). We have previ-ously shown that Tregs CD4þCD25þFoxp3þ are involved withmouse squamous cell carcinoma (SCC) development (5). How-

ever, the molecular basis dictating the homing of Tregs to thetumor microenvironment during SCC development is poorlyunderstood.

A complex chemokine–chemokine receptor interaction isinvolved with immune cell migration to tumor microenviron-ment (7). Chemokines produced in inflammatory tissues andlead to the accumulation of CCR5þ Tregs in tumor microenvi-ronment (2, 8). Recently, it was shown that CCL3, CCL4, andCCL5 secreted by tumor-infiltrating myeloid-derived suppressorcells recruit high numbers of Tregs favoring lymphoma growth(9). Murine Tregs expressing CCR5 respond to CCR5 chemokines(10) and CCR5 guides the homing of Tregs cells to Leishmaniamajor- and P. brasiliensis-infected tissues (11, 12). In addition,higher CCL5 production was associated with increased migrationof CCR5þ Tregs in a mouse model of pancreatic adenocarcinoma(3). The accumulation of CCR5þ cells in the tumor tissue pro-motes cancer angiogenesis, pulmonary metastasis, and protectscancer cells from antitumor immunity (13, 14).

These data prompted us to hypothesize that CCR5-dependentchemotaxis could be necessary for Treg migration into SCC, andtheCCR5 blockade could not only decrease the number of tumor-infiltrating Tregs but also improve antitumor immune response.Herein, using a murine SCC tumor model, we demonstrated thepresence of Treg cells in the SCC tumor microenvironment andthat CCR5 ligands, such as CCL4 and CCL5, were produced in thetumor. Moreover, absence of CCR5 resulted in decreased Tregsmigration to the tumor site, lower tumor numbers, and slowertumor progression. The adoptive transfer of CCR5þ Tregs resulted

1Department of Stomatology—Oral Pathology, Bauru School of Dentistry, Uni-versity of S~ao Paulo, Bauru, S~ao Paulo, Brazil. 2Department of BiologicalSciences, Bauru School of Dentistry, University of S~ao Paulo, Bauru, S~ao Paulo,Brazil. 3Department Research Division, Lauro de Souza Lima Institute, HealthState Department, Bauru, S~ao Paulo, Brazil. 4Department of Microbiology andImmunology, Institute of Biosciences of Botucatu, S~ao Paulo State University,Botucatu, S~ao Paulo, Brazil. 5Department of Biochemistry and Immunology,School of Medicine of Ribeir~ao Preto—University of S~ao Paulo, Ribeir~ao Preto,S~ao Paulo, Brazil. 6Samuel Oschin Comprehensive Cancer Institute, Cedars SinaiMedical Center, Los Angeles, California.

Corresponding Author: Ana Paula Campanelli, Bauru School of Dentistry,University of S~ao Paulo. Al. Dr. Oct�avio Pinheiro Brisolla, 9-75, CEP: 17012-901, Bauru, S~ao Paulo, Brazil. Phone: +55 14 3235-8271; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-0341

�2017 American Association for Cancer Research.

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in increased susceptibility to SCC development, whereas theadoptive transfer of conventional CD4þ T cells to CCR5-deficientmice induced the development of SCC that were moderatelydifferentiated. Furthermore, the adoptive transfer of CD8þ T cellsinhibited SCC development. When we assessed the developmentof lesions in CCR5-deficient animals after the adoptive transfer ofCD8þ T cells, we observed a significant inhibition of the progres-sion of papillomas to SCC, suggesting that themigration of T cellsto the tumor microenvironment is promoting tumor growth viainhibition of cytotoxic T cells. Thus, our study suggests that Tregmigration into the SCC tumor microenvironment is mediated byCCR5, and these cells are promoting tumor growth via inhibitionof antitumor cells such as cytotoxic CD8þ T cells. Our findingsreinforce the therapeutic potential of CCR5 inhibition for cancertreatment, and indicate an attractive approach for SCC treatment.

Materials and MethodsMice

Pathogen-free male CCR5-deficient (CCR5KO) and C57BL/6(wild type; WT) mice were provided by School of Medicine ofRibeir~ao Preto, University of S~ao Paulo (S~ao Paulo, Brazil).Experimental groups (n¼ 5)weremaintained at the Bauru Schoolof Dentistry, University of S~ao Paulo. Age and sex-matched micewere used for all experiments. Each mouse was housed in anisolated cage, containing sterilized feed, autoclaved bedding, andwater. All the animal experiments in this study were approved bythe Animal Research Ethics Committee of the Bauru School ofDentistry, University of S~ao Paulo (S~ao Paulo, Brazil).

Skin carcinogenesisFor two-stage carcinogenesis, mouse dorsal skin was shaved,

and treated once with DMBA (7,12-dimethylbenz[a]anthracene,Sigma-Aldrich; 125mg in 0.2mL acetone). After 1week,miceweretreated with PMA (Phorbol 12-myristate 13-acetate, Sigma-Aldrich; 10 mg in 0.1mL acetone) three times weekly for 35 weeks(15). Mice were examined weekly. Growths >1 mm diameter,persisting for �2 weeks were recorded as tumors. Survival rateswere determined in independent groups of animals. Tumors werecollected and analyzed at 35 weeks after initiation.

Adoptive transfer of CCR5þCD4þCD25þ, CCR5þCD4þCD25�,or CCR5þCD8þ T cells

CD4þ lymphocytes were enriched from the WT mouse spleenusing the IMag Cell Separation System [MACS isolation kitaccording to the manufacturer's instructions (Miltenyi Biotec)].CD8þ lymphocytes were isolated from the WT mouse spleenusing the Mouse CD8 Cell Enrichment Set-DM IMag IsolationKit according to themanufacturer's instructions (BDBiosciences).T-cell subsets CD4þCD25�, CD4þCD25þ or CD8þ (all in theamount of 2 � 105 cells) were intravenously transferred intoCCR5KO mice at 5 weeks after skin carcinogenesis initiation.

Isolation of leukocytes from tumor samplesThe protocol used to isolate leukocytes from the tumor micro-

environment has been described previously (16). The tissuehomogenates were filtered using a 40-mm cell strainer (Falcon;BD Biosciences), the isolated cells were prepared for flow cyto-metry analysis, and the supernatants of the tumor samples werestored in protease inhibitor solution (Roche) at�20�C until usedto measure cytokine level.

Flow cytometryFor immunostaining, FcgRswere blockedwithmAb2.4G2, and

the cells were then stained for surface markers using PerCP-, PE-,and FITC-conjugated antibodies against CD3 (145-2C11), CD4(RM4-5), CD8 (53-6.7and 53-6.7), CD19 (1D3), DC (33D1),F4/80 (BM8), CD11b (M1/70), CD11c (HL3), CD14 (rmC5-3),CD25 (PC61), CD18 (3.751), CD45RA (148), CD45RB (16A),CD62L (MEL-14), CD69 (H1.2F3), CD95 (Jo2), CD103 (M290),CD154 (MR1), CD178 (MFL3), LAG3 (C9B7W), CCR5 (C34-3448), NK1.1 (PK136), GITR (DTA-1), Gr1 (RB6-8C5), and therespective goat or rat isotype controls were used (BDBiosciences).The intracellular detection of Foxp3 (MF23) and CCL4 (A65-2;BD Biosciences) in leukocytes was performed using Cytofix/Cytoperm and Perm/Wash buffer from BD Biosciences accordingto themanufacturer's instructions. The data were collected using aFACSCalibur flow cytometer (BD Immunocytometry Systems)and analyzed using CellQuest software (BD Biosciences).

Histopathologic analysisFor histologic analysis, excised skin samples were fixed with

10% buffered formalin and processed using routine histologictechniques. Longitudinal sections of 5-mm-thick were stainedwith hematoxylin and eosin (H&E) and analyzed by a pathologistblinded to the experimental groups. All sections were analyzedunder an optical microscope, and microphotographs were col-lected using a digital camera (Leica DFC310 FX, Leica Microsys-tems GmbH).

Cytokines and chemokinesThe concentrations of IL10, IL12, IFNg , TNFa, TGFb, IL17,

CCL4, CCL5, CCL17, and CCL22 in the supernatants of tumorsamples were measured using mouse ELISA kits (BD Biosciencesor R&D Systems), according to the manufacturer's instructions.

Statistical analysisThe unpaired Student t test and ANOVAwas used for statistical

comparisons ofmultiple groups. Log-rank (Mantel–Cox) test wasused to determine whether the CCR5-deficient mice had betteroutcomes than the wild-type mice. P values �0.05 were consid-ered statistically significant.

ResultsAbsence of CCR5 affects SCC development

High number of the CCR5þ cells in the tumor microenviron-ment has been described previously (3, 17–20). So we firstassessed CCR5 expression on T cells in the SCC tumor. Foxp3þ

T cells from wild-type (WT) mice with SCC displayed high levelsof CCR5 expression (Fig. 1A). To investigate the role of the CCR5during SCC development, CCR5-deficient (CCR5KO) and WTmice were submitted to a multistage tumorigenesis protocol. Aspreviously described, this method results in the development ofvisible tumors on WT mice at 8 weeks after carcinogenic induc-tion. Our results showed that the SCC development was delayedin CCR5KO mice (Fig. 1B). Also, the average number and size oftumors per mouse also was significantly reduced in the CCR5KOmice (Fig. 1C). CCR5KO mice developed lesions with lowervolumewhen comparedwithwild-typemice (Fig. 1D).Histologicexamination revealed that all WT mice included in the studydeveloped high-grade dysplasia, of which 100%were classified as

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SCC invasive well differentiated, while only 44% of the CCR5KOmice developed SCC in situ (Fig. 1E and F).

Absence of CCR5 reduces Tregs accumulation on the tumormicroenvironment

We next investigate whether CCR5 is involved in the recruit-ment of tumor-infiltrating leucocytes. Enumeration of the isolat-ed cells from WT and CCR5KO tumors showed a significantincrease in the absolute numbers of total leukocytes in CCR5KOmice when compared with WT mice (Fig. 2A). Importantly, theabsolute number of CD4þ T, B, and NK cells was significantlyhigher in the tumors of CCR5KO mice (Fig. 2). However, infil-tration of CD8þ T cells was significantly lowered in tumors ofCCR5KO mice (Fig. 2C). The absolute number of macrophages(F4/80þ CD11bþ) and dendritic cells (CD11cþ CD11bþ) wassimilar in CCR5KO and WT mice (Fig. 2E and F). Next, we

questionedwhether the absence ofCCR5would result in a specificalteration on the numbers of Tregs in the tumor microenviron-ment and tumor-draining lymph nodes (LN; Fig. 2I and J). Ourresults revealed that the absence of CCR5 resulted in a significantreduction in FoxP3þCD4þ T cells in the tumors (Fig. 2I). On theother hand, higher number of Tregs was found in the tumor-draining lymph nodes of CCR5KOmice than control group (Fig.2J). On the basis of these findings, CCR5 plays an important rolein the tumor-infiltrating lymphocytes during SCC development,particularly the infiltration of Tregs.

Adoptive transfer of Tregs or conventional CD4þ T to theCCR5-deficient mice enhanced SCC development

The results showing that CCR5-deficient mice presented sig-nificantly lower numbers of Tregs in SCC tumor, led us toinvestigate the direct effect of the migration of CCR5þ Tregs,

Figure 1.

Absence ofCCR5affects SCCdevelopment.CCR5KO (full squares) and WT (emptysquares) mice were treated according to achemical carcinogenic protocol usingDMBA and PMA for 35 weeks. A, Tumorsample from WT mice were collected andleukocytes were analyzed by flowcytometry after staining for Treg markersCD4, CD25, CCR5, and FoxP3. Dot plotshow the percentage of CCR5þFoxP3þ

cells. B, Kaplan–Meier curves showingtumor-free of wild-type (WT) and CCR5KOmice. The average number (C) and tumorsize (D) was measured. Data shownrepresent mean � SEM. E, H&E-stainedsections were scored to grade their level ofdysplasia. F, Representative pictures areshown of H&E-stained skin sections thatwere obtained from WT and CCR5KO miceat 35 weeks after SCC induction; scale bars,50 mm (top), 100 mm (bottom). The datarepresent the mean � SEM from threeindependent experiments, each with n ¼ 5;� , P � 0.05, compared with WT mice.

CCR5-Dependent Homing of Tregs Contributes to SCC Development

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conventional CD4þ and CD8þ T cells during SCC development.For these, we adoptively transferred CCR5-expressing Tregs, con-ventional CD4þ and CD8þ T cells fromWT-na€�vemice to DMBA-

treated CCR5KO mice. After 35 weeks, the transfer of Treg cellsinto CCR5KO mice resulted to a significant increase in tumorburden and incidence as compared with CCR5KO mice

Figure 2.

Absence of CCR5 reduces Tregs accumulation in the tumor microenvironment. A, The absolute number and phenotype of leukocytes in the tumor lesionswere determined by flow cytometry. Flow cytometry analysis of CD3þCD4þ T cells (B), CD3þCD8þ T cells (C), B cells (D), macrophages (E), dendritic cells (F),myeloid cells (G), and NK cells (H). The absolute number of CD4þFoxP3þ T cells in tumor microenvironment (I) and lymph node (J). Values are mean �SEM; �, P � 0.05, compared with sham mice; #, P � 0.05, compared with WT mice.

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littermates (Fig. 3AandB). Visible tumorsweredetectedat 8weeksafter carcinogenic induction, whereas no tumors were detected inCCR5KO littermates (Fig. 3A and B). Interestingly, while thetransfer of CD4þ T to CCR5KO mice led to early incidence ofpapilloma and tumor development, the adoptive transfer ofCD8þ T cells resulted in similar tumor burden and incidencecompared with CCR5KO littermates (Fig. 3A). Histologic exam-ination revealed that 75% of the mice that received Tregs devel-oped invasive SCC that was well differentiated (Fig. 3C and D).Moreover, 66% of the mice receiving conventional CD4þ T-cellreceptors developed invasive andwell-differentiated SCC (Fig. 3Cand D). All CCR5KOmice receiving CD8þ T cells developed onlypapillomatous lesions, presenting epithelial hyperplasia andmildepithelial dysplasia (confined to the basal and parabasal layers ofthe epithelium) and a moderate inflammatory infiltrate in theunderlying connective tissue (Fig. 3C and D).

Adoptive transfer of Tregs into CCR5-deficient mice increasedCCL4-producing T-cell accumulation in the tumormicroenvironment

To address the effects of Tregs, CD4þ, and CD8þ T-cell transferto DMBA-treated CCR5KO mice, we next analyzed the tumor-infiltrating cells. The results revealed that transfer of Tregs orconventional CD4þ T cells resulted in less infiltration of inflam-matory leukocytes (Fig. 4A), whereas higher leukocyte infiltrationwas observed after CD8þ T-cell transfer into CCR5KO mice. Thetransfer of Tregs or conventional CD8 T cells was accompanied bypredominant infiltration of CD8T cells (Fig. 4C), B cells (Fig. 4D),macrophages (Fig. 4E), dendritic cells (Fig. 4F), myeloid suppres-sor cells (Gr1þCD11bþ cells; Fig. 4G), NK (Fig. 4H), andCD4þFoxp3þ (Treg) cells (Fig. 4I) compared with CCR5KOmice.Recently, it was described that CCL3/CCL4–CCR5 pathway is thepredominant pathway that mediates the ability of Tregs to attract

Figure 3.

Adoptive transfer of Tregs and conventional CD4þ T cells into CCR5-deficient mice enhanced SCC development. CCR5-deficient mice adoptively transferredwith Tregs, CD4þ, or CD8þ T cells were analyzed at 35 weeks after SCC induction. Controls CCR5-deficient mice received PBS vehicle alone (vec). A, Theaverage number of tumorswasmeasured. Data shown representmean� SEM; � , P�0.05, comparedwith CCR5KOmice.B,Kaplan–Meier curve showing tumor-freemice. C, H&E-stained sections were scored for grading of dysplasia. D, Representative images showing dysplasia in skin tissue; scale bars, 50 mm (top),100 mm (bottom). Representative data are show from at least three independent experiments, each with n ¼ 5.






CD4 and CD8 T cells (21). In addition, CCL4 production byconventional CD4 T cells is critical for the development of T-cellmemory (22). Interestingly, the adoptive transfer of Tregsincreased the absolute numbers of CD4þ and CD8þ T cellscoexpressing CCL4 in the tumor microenvironment. Similarresults were observed after transfer of conventional CD4þ T cells,except in the numbers of CD8þCCL4þ cells (Fig. 4J and L).

Transfer of CD8þ T cells improved Th1-related immuneresponse in the tumor microenvironment

To assess whether the modification of inflammatory cellinfiltrate observed in CCR5KO mice after the adoptive transferof Tregs, CD4þ, and CD8þ T cells would affect subsequentinflammatory cytokine/chemokine profile, we evaluated IL12(p40), IFNg , TNFa, IL17, IL10, and TGFb levels in the tumormicroenvironment.

The presence of tumors inWT and CCR5KOmice increased thelevels of TNFa, IFNg , IL12, IL10, and CCL5 (Fig. 5). CCL22 andCCL4 levels were significant decreased in CCR5KO mice when

compared with WTmice (Fig. 5H and J). IL17 levels were higherin tumors from CCR5KO mice compared with WT mice (Fig.5E). Similar levels of TGFb were detected in samples fromCCR5KO and WT mice (Fig. 5D). Consistent with the enhancedinfiltration of leukocytes after the transfer of Tregs, we found anelevated level of TNFa (Fig. 5A), TGFb (Fig. 5D), IL17 (Fig. 5E),IL10 (Fig. 5F), CCL4 (Fig. 5H), CCL17 (Fig. 5I), and CCL22(Fig. 5J) in the tumor samples from CCR5KOmice that receivedTregs. The analysis of cytokines and chemokines after CD4þ

T-cell transfer to CCR5KO animals revealed that the levels ofIFNg , TNFa, IL12, TGFb, IL17, IL10, CCL5, and CCL22 werefound to be elevated in the tumor microenvironment, com-pared with CCR5KO mice. Notably, as a result of the antitumoreffect of CD8þ T-cell transfer in CCR5KO mice, these cellsgenerate an immune response characterized by higher levelsof IFNg , IL12, and TNFa. In contrast, the transfer of Tregs orCD4þ T cells to CCR5KO mice induced a mixed inflammatoryand anti-inflammatory immune response, which seems tocontribute to an uncontrolled tumor growth.

Figure 4.

Characterization of leukocyte populations in the tumor microenvironment after adoptive transfer of Tregs, CD4þ and CD8þ T cells into CCR5-deficient mice.A, The absolute number of leukocytes. Bars show the frequencies of CD3þCD4þ (B), CD3þCD8þ (C), B cells (D), macrophages (E), DCs (F), myeloid cells (G), NK (H),CD4þFoxp3þ (I), CD4þCCL4þ (J), CD8þFoxp3þ (K), and CD8þCCL4þ (L) cells in the tumor. The data are representative of three experiments; �, P � 0.05compared with CCR5KO mice.

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DiscussionBecause themigration of Tregs to the tumormicroenvironment

is related to tumor escape mechanisms, altering and/or blockingthe migration of these cells is an exciting field to be explored inimmunotherapies. Tregs express high levels of CCR5 and theexpression of this receptor leads to an increased susceptibility tochemical carcinogenesis. In fact, CCR5KO mice exhibited signif-

icant less tumor incidence and a Th1-biased response character-ized by increased IL12 and lower IL10 levels. Cytokine analysisalso revealed that CCL4, CCL17, and CCL22 levels decreased inthe tumors of CCR5KOmice. The analysis of leukocytes infiltratein the tumormicroenvironment revealed that Tregs were detectedin lower numbers in the skin of CCR5KO mice, when comparedwith WT mice. It is already well established the importance thatTregs play in the tumor development and progression (23) and

Figure 5.

Adoptive transfer of CD8 T cells improved Th1-related immune response in the tumor microenvironment. TNFa (A), IFNg (B), IL12 (C), TGFb (D), IL17 (E), IL10 (F),CCL5 (G), CCL4 (H), CCL17 (I), and CCLL22 (J) levels were measured in supernatants of tumor samples using ELISA. The data are shown as the mean � SEMand are representative of at least three independent experiments; � , P � 0.05 compared with CCR5KO mice.






the CCR5-dependent chemotaxis of Tregs to tumor microenvi-ronment (3). Consistent with this, we observed no malignanttransformation of papillomas to invasive carcinoma in CCR5KOmice. In accordance with these data, other studies showed tumorgrowth might be inhibited by CCR5-mediated blockade of Tregmigration without affecting the migration of effector T cells tolesions (3). Collectively, these results suggest that themigration ofTregs to the tumormicroenvironment is dependent on CCR5 andCCR5þ Tregs favoring the development of SCC.

To confirm the association between higher susceptibility toSCC development with the Treg chemotaxis via CCR5 pathway,we evaluated the development of SCC in CCR5-deficient miceanimals undergoing adoptive transfer of CCR5þ Tregs. Transfer ofTregs to CCR5KO mice increased tumor numbers and malignanttransformation (papilloma to undifferentiated invasive carcino-ma), thatwas associatedwith increased infiltrationof Tregs aswellas decreased infiltration of NK cells, thus rendering these micemore susceptible to tumor development. These data suggest thatCCR5-dependent chemotaxis of Treg cells may have an indirecteffect on cancer progression by controlling the antitumor immuneresponses. Increasing evidence suggest that CCR5þ Tregs exert finecontrol on themagnitude of the antitumor immune response, andthese cellsmight negatively influence the activation and/ormigra-tion of cytotoxic CD8 T lymphocytes, facilitating tumor develop-ment (24–26). In fact, recruitment of CD8þ T cells decreased inthe tumors of CCR5KOmice. However, although CCR5KO groupshowed decreased accumulation of CD8þ T cells in the tumormicroenvironment, the trafficking in CCR5KO mice could alsooccur independently of CCR5 (27). Probably others receptor–ligand interactions could influence CD8þ T-cell migration. AsCCL4, CCL17, and CCL22 levels decreased in CCR5KO micetumors, we might speculate, then, that the lack of these chemo-kines could be associated with limited infiltration of CD8þ T cellsin CCR5KO mice. In fact, increased production of CCL4 andCCL22was observed in lesions fromCCR5KOmice after adoptivetransfer of Tregs or CD4þ T cells, associated with increased inCD8þ T-cell infiltration. Also, other chemokines (CCL2, CXCL9,and CXCL10) could be also involved (28).

The evaluation of inflammatory mediators correlated with thesuppression of antitumor immune responses by the presence ofCCR5þ Tregs. Like WT mice, increased production of CCL4 andCCL22was observed in lesions fromCCR5KOmice after adoptivetransfer of Tregs, associated with increased infiltration of CD8þ Tcells. The production of CCL4 represents an additional immu-nosuppressive mechanism that Tregs use to attract CCR5-expres-sing CD4 and CD8 T cells to facilitate their proximity-dependentmechanisms of action (29). In addition, high production of TNFaand IL17 in the tumor microenvironment was observed. Highproduction of IL17 has been reported in different types of tumors(30, 31). Moreover, a recent study shows that Tregs produce IL17and promote the earliest stages of colon carcinogenesis (31). Theprogression of tumors that were chemically induced has also beenassociated with the higher frequency of myeloid-suppressive cellsand reduced infiltration of CD8 T cells induced by IL17 (32). Inaddition, higher production of IL10 and TGFb in lesions afteradoptive transfer of Tregs couldbe correlatedwith the suppressionof antitumor immune response, and may be produced by theexpansion of Tregs in the tumor microenvironment after thetransfer. TGFb inhibits the proliferation of effector T cells andmacrophage activation that may contribute to the tumor devel-opment (33–35). TGFb not only contributes to the growth of

tumor heterogeneity at early stages of tumorigenesis, inducing thegeneration of a TGFb-responding progeny that shows aberrantdifferentiation, increased invasiveness, and drug resistance butalso induces malignant conversion of papillomas to SCCs andmetastasis at latter stages (36). In addition, some studies haveshown that TGFb and IL10 create a favorable environment for theconversion of conventional Foxp3� T cells in Foxp3þ Tregscapable of suppressing antitumor immune response (34, 37–41). These findings suggest that in SCC tumor microenvironmentthe conversion of conventional CD4þ T cells in induced Tregs(iTreg) might contribute to tumor progression. In contrast, Th1cells have been explored as most efficient effector T cells subset inantitumor immunity. Previous study shows that Th1 lymphocytesexpress CXCR3, CCR1, and CCR5, whereas Th2 lymphocytesexpress CCR8, CXCR4, CCR3, and CCR4 (41). To obtain addi-tional insights into SCC development in CCR5-deficient mice, wealso transfer conventional CD4þ T to CCR5KO mice. The char-acterizationof the inflammatory infiltrate showed that onlyCD4þ

T cells were detected in higher proportions in the lesions of theseanimals. Here we demonstrated that the transfer of na€�ve CD4þ Tcells to CCR5-deficient mice increased susceptibility to chemicalcarcinogenesis, suggesting that the tumor is modeling the phe-notype of these cells, possibly via the differentiation of Tregs orTh2 subsets. This is an area of further investigation. In addition,lower number of NK cells was observed in the lesions comparedwith CCR5-deficient mice. Such evidence points to a role ofconventional CD4þ T cells in the inhibition of the activationand/or migration of NK cells resulting in increased susceptibilityof these animals to chemical carcinogenesis.

It is becoming clearer that the development and/or mainte-nance of effective antitumor immune response depend on CD8þ

T-cell activation (42). The transfer of CD8þ-na€�ve T cells toCCR5KO mice decreased not only number of tumors but alsopapilloma-malignant conversion and favored the infiltration ofCD8þ T lymphocytes and NK cells besides inhibiting Tregs,rendering these mice less susceptible to tumor development.These mice exhibit Th1-biased immune responses with increasedIL12, IFNg , and TNFa production that would contribute to theestablishment of an effective immune response to control tumordevelopment. In fact, we showed previously that higher levels ofIFNg and lower levels of TGFb in the tumor microenvironmentfrom anti-PD-1–treated mice might provide an explanation forthe control of tumor development (43). Furthermore, in anindirect manner, lower levels of TGFb observed after adoptivetransfer of CD8þ T cells would also inhibit cellular clonal het-erogeneity expansion previously associated with increased inva-siveness and metastatic abilities (36). The presence of IL12 isrelated to the development of Th1 responses, via IFNg and TNFaproduction that stimulates cytotoxic activity of CD8 T cells andNK cells, induces M1 polarization of macrophages, and inhibitsangiogenesis promoting the development of effective antitumorimmune response (35, 44–47). Although cytotoxic TCD8andNKcells constitute two major antitumor cell populations, and theimpairment of their activation has been correlated with tumorprogression (48, 49), we have not found any evidence of anincreased cytotoxic activity of splenic NK cells in all groupsanalyzed. However, we cannot exclude that the cytotoxic activityof NK cells are not systemic but restricted to the tumor environ-ment. Our data identified that the higher number of CD8 Tlymphocytes accompanied by a low frequency of Tregs in thelesionswere associatedwith abetter immune response, suggesting

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that the low frequencies of Tregs contributes to the less suscep-tibility of these animals to the development of SCC.

Altogether, our results strongly suggest that, during SCC devel-opment, unknown factors released from tumor cells, stroma, oreven immune cells possibly recruit Tregs via a specific subset ofchemokines and CCR5 receptors to modulate antitumor immu-nity. A correlation between chemotaxis Tregs via CCR5 and SCCtumor development and progression were established, showingthat these cells regulate critical aspects of this disease and opennew perspectives of an immunotherapy target.

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

Authors' ContributionsConception and design: G.P. Garlet, J.S. Silva, A.P. CampanelliDevelopment of methodology: C.E. de Oliveira, M.R.S. Nogueira, V.S. Lara,T.H. Gasparoto, C.R. Pinheiro, J.S. da SilvaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Kaneno, G.P. Garlet, A.P. CampanelliAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.E. de Oliveira, T.H. Gasparoto, C.R. Pinheiro,N.G. Amor

Writing, review, and/or revision of the manuscript: C.E. de Oliveira,K.A. Cavassani, A.P. CampanelliAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.S. Silva, A.P. CampanelliStudy supervision: A.P. CampanelliOther (experimental investigation): T.H. Gasparoto

AcknowledgmentsWe thank A. Silva for excellent technical assistance.This work was supported by Fundac~ao de Amparo �a Pesquisa do Estado de

S~ao Paulo - FAPESP (grant no. 2012/15331-0), Coordenac~ao de Aper-feicoamento de Pessoal de Nível Superior (CAPES scholarship; to C.E. deOliveira and C.R. Pinheiro), and Conselho Nacional de DesenvolvimentoCientífico e Tecnol�ogico (CNPq scholarship; to J.S. Silva, G.P. Garlet, andA.P. Campanelli).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 24, 2017; revised August 14, 2017; accepted September 7,2017; published OnlineFirst September 13, 2017.

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de Oliveira et al.




Published OnlineFirst September 13, 2017.Mol Cancer Ther Carine Ervolino de Oliveira, Thaís Helena Gasparoto, Claudia Ramos Pinheiro, et al. Carcinoma DevelopmentMicroenvironment Contributes to Skin Squamous Cell CCR5-Dependent Homing of T Regulatory Cells to the Tumor

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