Melanoma Induces, and Adenosine Suppresses, CXCR3 ... · Eleanor Clancy-Thompson1,2,Thomas J....

Research Article

Melanoma Induces, and Adenosine Suppresses,CXCR3-CognateChemokineProductionandT-cellInfiltration of Lungs Bearing Metastatic-likeDiseaseEleanor Clancy-Thompson1,2, Thomas J. Perekslis1,2,Walburga Croteau3, Matthew P.Alexander1,2,Tamer B.Chabanet1,2, Mary Jo Turk1,2,YinaH. Huang1,3, andDavidW.Mullins1,2

Abstract

Despite immunogenicity, melanoma-specific vaccines havedemonstrated minimal clinical efficacy in patients with estab-lished disease but enhanced survival when administered in theadjuvant setting. Therefore, we hypothesized that organs bearingmetastatic-like melanoma may differentially produce T-cell che-motactic proteins over the course of tumor development. Usingan established model of metastatic-like melanoma in lungs, weassessed theproductionof specific cytokines and chemokines overa time course of tumor growth, and we correlated chemokineproduction with chemokine receptor–specific T-cell infiltration.We observed that the interferon (IFN)-inducible CXCR3-cognatechemokines (CXCL9 and CXCL10) were significantly increased inlungs bearing minimal metastatic lesions, but chemokineproduction was at or below basal levels in lungs withsubstantial disease. Chemokine production was correlated

with infiltration of the organ compartment by adoptivelytransferred CD8þ tumor antigen-specific T cells in a CXCR3-and host IFNg-dependent manner. Adenosine signaling in thetumor microenvironment (TME) suppressed chemokine pro-duction and T-cell infiltration in the advanced metastaticlesions, and this suppression could be partially reversed byadministration of the adenosine receptor antagonist aminoph-ylline. Collectively, our data demonstrate that CXCR3-cognateligand expression is required for efficient T-cell access oftumor-infiltrated lungs, and these ligands are expressed in atemporally restricted pattern that is governed, in part, byadenosine. Therefore, pharmacologic modulation of adeno-sine activity in the TME could impart therapeutic efficacy toimmunogenic but clinically ineffective vaccine platforms.Cancer Immunol Res; 3(8); 956–67. �2015 AACR.

IntroductionActive vaccination has proven immunogenic (1), increasing the

number of circulating tumor antigen-specific CD8þ effector ormemory T cells. However, therapeutic vaccination imparts min-imal clinical efficacy against active disease (2), even though ex vivoanalyses demonstrate that vaccine-induced cytotoxic T lympho-cytes lyse tumor targets in vitro (3). The failure of vaccines in thecontext of active disease may be ascribed to tumor-induced localimmune suppression, myeloid-derived suppressor cells (MDSC)or regulatory T-cell (Treg) activity, or tumor immune editing to

evade T-cell recognition (4). However, T-cell access of tumors isassociatedwith improved prognosis in primary (5) andmetastatic(6) melanomas, yet multiple studies have demonstrated thateffector T-cell infiltration of primary or metastatic melanomas isvariable and often absent. Thus, the efficacy of vaccination likelyrelies upon efficient infiltration of tumors by treatment-inducedantigen-specific T cells.

Circulating effector T cells access peripheral tissues, includingtumors and tumor-infiltrated organs, through the engagementof integrins with their ligands on the vascular endothelium;firm adhesion and extravasation of T cells require high-affinityintegrin binding, which is activated by chemokine receptor(CCR) in response to specific ligands (chemokines). Recentstudies have highlighted the clinical importance of chemokineexpression for T-cell infiltration into the tumor microenviron-ment (TME; ref. 7). Likewise, expression of CXCR3-cognatechemokines (CXCL9, CXCL10, and CXCL11) is correlated withT-cell infiltration status in metastatic melanoma (8). Previous-ly, we demonstrated that melanoma cells from lymph nodemetastases are capable of producing CXCR3-cognate chemo-kines following stimulation with interferon (IFN)-a or IFNg(9). Furthermore, the presence of tumor-specific CD8 cellsexpressing CXCR3 was correlated with the duration of survival(10). Collectively, these observations suggest an important rolefor the CXCR3 chemotactic axis for the temporal and spatialtargeting of circulating T effector cells to compartments withmelanoma metastases.

1Department of Microbiology and Immunology, Geisel School of Med-icine at Dartmouth, Lebanon, New Hampshire. 2Norris Cotton CancerCenter,Geisel School ofMedicine atDartmouth, Lebanon,NewHamp-shire. 3Department of Pathology, Geisel School of Medicine at Dart-mouth, Lebanon, New Hampshire.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Current address for E. Clancy-Thompson: Dana-Farber Cancer Institute, 450Brookline Avenue, Boston, Massachusetts.

Corresponding Author: David W. Mullins, Norris Cotton Cancer Center, GeiselSchool of Medicine at Dartmouth, 1 Medical Center Drive, HB7937, Lebanon,NH 03756. Phone: 603-653-9922; Fax: 603-653-9952; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-15-0015

�2015 American Association for Cancer Research.

CancerImmunologyResearch

Cancer Immunol Res; 3(8) August 2015956

on September 5, 2020. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst June 5, 2015; DOI: 10.1158/2326-6066.CIR-15-0015

http://cancerimmunolres.aacrjournals.org/

Although vaccination has shown minimal efficacy for treat-ment of advanced disease, adjuvant-setting vaccination has pro-longed disease-free survival (11), suggesting that newly formingmetastatic lesions may be receptive to T-cell infiltration, and byextension, replete with chemokines; in contrast, more advancedand established tumors may lack T-cell infiltration as a conse-quence of suppressed chemokine production. To date, no studyhas evaluated the possibility of differential chemokine produc-tion and T-cell infiltration over the course of tumor growth.

Using an established murine model of metastatic-like mela-noma in the lungs, we demonstrate differential regulation ofCXCR3-cognate chemokine and infiltration by CXCR3þCD8þ Tcells in early- versus advanced-stage tumors. Importantly, T-cellinfiltration of advanced tumors can be partially restored throughblockade of adenosine receptor signaling. Thus, we demonstrate adeficit in immune therapy of cancer and a potential means toenhance the antitumor efficacy of vaccine or adoptive T cell–mediated therapy of established metastatic disease.

Materials and MethodsTumor models

Metastatic-like tumors were established in C57BL/6 mice (TheJackson Laboratory strain 00664), Rag�/� (The Jackson Labora-tory strain 002216), or IFNg�/� (The Jackson Laboratory strain002287), as indicated, by i.v. injection of 3 � 105 B16-F10 (B16)melanoma cells. B16 cells were obtained from the ATCC (CRL-6475), and tumors were established from cryopreserved stocksthat had been passaged less than two times. Tumor developmentwas observed in 100% of injected mice.

Inducible melanomas were established by intradermal injec-tion of 4-hydroxy-tamoxifen (10 mL of a 20-mmol/L solution inDMSO) in Tyr::CreERþ/�; BrafCA/þ; Ptenlox/lox mice (12) that hadbeen backcrossed onto a B6 background, as described previously(13).

For all studies, animals were maintained in pathogen-freefacilities at the Geisel School of Medicine (Lebanon, NH), andall procedures were approved by the Dartmouth College Institu-tional Animal Care and Use Committee.

Cytokine and chemokine production analysesProtein from perfused lungs was isolated using Total Protein

Extraction Reagent (Thermo Scientific) and cOmplete proteaseinhibitor (Roche cOmplete). Mouse CXCL9, CXCL10, and IFNgELISA (DuoSets) were obtained from R&D Systems and per-formed according to the manufacturer's protocol.

Gene expression analysesTotal RNA from cultured B16 melanoma cells, perfused lungs,

or Braf/Pten tumors was isolated (Qiagen RNeasy kit) and thentranslated into cDNA using a High Capacity RNA-to-cDNA kit(Applied Biosystems). Specific gene expression was quantified byqPCR using master mix and prevalidated gene-specific primers(Life Technologies) and a StepOne Plus instrument (AppliedBiosystems).

Lung sections and immunohistochemistryMice were perfused with 4% paraformaldehyde (PFA; Sigma-

Aldrich) prior to lung dissection. Lungs were sequentially incu-bated for 1 hour at 4�C in (i) 4%PFA, (ii) 30% sucrose in PBS, and(iii) 15% sucrose, 50% optimum cutting temperature (OCT;

Tissue Tek, Sakura) in PBS followed by embedding in OCT.Ten-micrometer sections were performed with a cryostat, fixedin cold acetone, rehydrated in PBS, and blocked in 5%BSA in PBS.Sections were stained overnight with primary antibodies againstCXCL9, CXCL10 (rabbit, Bioss), and TA99 (ATCC clone HB-8704), followed by Alexa Fluor 594- and Alexa Fluor 647–con-jugated secondary antibodies againstmouse and rabbit IgG in 1%normal goat serum (Jackson ImmunoResearch and Life Technol-ogies), for 1 hour at room temperature. Microscopy was per-formed on a Zeiss AX10 fluorescence microscope fitted with aCoolSnap HQ2 mono14-bit camera, using a �20 Apo objective.Images were taken with ZEN Pro 2012 (Zeiss) software andprocessed with Fiji image processing software.

ReagentsAntibodies specific for murine CD183 (CXCR3, clone CXCR3-

173), CXCL9 (clone MIG 2F5.5), IFNg (clone XMG1.2), CD4(clone RM4-5), CD8a (clone 5H10-1), CD45 (clone 30-F11),CD11c (cloneN418), Ly6G (clone 1A8), CD335 ([NKp46], clone29A1.4), CD90.1 (Thy1.1, clone OX-7), CD31 (clone 390), andCD16/32 (clone 93) were obtained from BioLegend. Anti-aden-osine A2a receptor (ADORA2A; clone 7F6-G5-A2) was obtainedfrom Novus Biologicals.

Adoptive transfer modelTumors were allowed to establish for 3, 11, or 18 days before

adoptive transfer of T cells. T cells for adoptive transfer werecollected from spleens of T-cell receptor (TCR) transgenic donormice. To facilitate tracking of transferred cells, specific TCR micewere crossed onto a Thy1.1 x Rag�/� background (The JacksonLaboratory strains 000406 and 002216, respectively). To ensuretumor-antigen specificity, T cells specific for an endogenousmelanocyte/melanoma antigen (gp100) were obtained fromPmel� Thy1.1 x Rag�/� mice (crossed from Pmel TCR mice; TheJacksonLaboratory strain005023; henceforth termedPmelmice);thesemice were then crossed onto a CXCR3-deficient backgroundand henceforth termed Pmel/CXCR3�/� mice.

Before transfer, TCR transgenic CD8 T cells were enriched usinga MACs negative selection magnetic kit (Miltenyi Biotec), thenactivated by culture (37�C/5%CO2 for 24 hours) with anti-CD3and anti-CD28 Dynabeads (Life Technologies) at a 1:1 ratio. Foradoptive transfer studies, 1� 106 in vitro–activated CD8þ Pmel orPmel/CXCR3�/� cells were i.v. injected intomice at various stagesof tumor growth, as indicated, then recovered and enumerated 1hour later (14).

To assess T-cell infiltration of tumor-bearing lungs, the lungswere perfused with 1 mL 1� PBS and homogenized to single-cellsuspensions and enriched for lymphocytes using a gentleMACStissue dissociator and lung dissociation kit (Miltenyi Biotec).Magnitude of T-cell infiltration was assessed by multi-color flowcytometry (MACSQuant, Miltenyi Biotec). Monoclonal antibo-dies against CD8, CD45, CXCR3, and Thy1.1 (BioLegend) wereused to track adoptively transferred T cells, and viability wasconfirmed with violet viability dye (Life Technologies).

Flow cytometric staining and analysesFor surface staining, cells were suspended in FACS buffer (1%

BSA in PBS) and incubated with purified antibodies for 30minutes at 4�C, then washed three times in FACS buffer andfixed in 0.5% PFA. For intracellular staining, cells were stained for

Adenosine Suppresses Melanoma-Induced CXCR3 Chemokines

www.aacrjournals.org Cancer Immunol Res; 3(8) August 2015 957




surface markers and fixed with a 1:4 dilution of Fixation Buffer(BioLegend) in PBS for 20 minutes; washed twice with a 1:10dilution of Permeabilization Buffer (BioLegend) in PBS (PermBuffer); incubated in Perm Buffer for 20 minutes; stained forCXCL9 or IFNg in Perm Buffer for 30 minutes; and washedtwice with FACS buffer. All data were acquired with a MiltenyiMacsQuant cytometer and analyzed with FlowJo (TreeStar)software.

In vitro migration studiesTo assess the migration potential of CD8þ T cells from Pmel

and Pmel/CXCR3�/� mice, magnetically enriched CD8 T cells(Miltenyi Biotec) were activated, as described above, and plated at5 � 104 cells into Transwell assay plates with 5 mmol/L mem-branes (Costar). Chemokines (CXCL10 or CCL5; R&D Systems)were added to the bottom wells, and CCR-specific blockingantibody (anti-CXCR3, BioLegend CXCR3-173) added to the topwells, as indicated. After 3-hour incubation at 37�C,migrated cellswere enumerated using an automated cell counter (Bio-RadTC10).

Aminophylline treatmentTo assess the role of adenosine-mediated signaling on cytokine

and chemokine expression and production in the TME, tumor-bearingmicewere treatedwith the nonspecific adenosine receptorantagonist aminophylline (theophylline ethylenediamine; Sig-ma-Aldrich) by i.p. injection of 50 mg/mL of drug every 3 days for18 days, as described previously (15).

In vitro tumor studiesB16melanomas were plated at 1� 105 cells per well in 24-well

tissue culture plates (Corning) at 37�C/5% CO2. After 24 hours,the supernatant was replaced and amended with IFNg (0, 500,2,500, or 5,000 pg; BioLegend), adenosine (ADO; 0, 500, or 1,000mmol/L, Tocris), or the adenosine A2 receptor–specific inhibitorZM241,385 (ZM; 0 mmol/L, 10 mmol/L; R&D Systems). After 24hours at 37�C/5% CO2, the supernatant was collected andCXCL10 protein production measured by ELISA.

ResultsDifferential CXCR3-cognate chemokine production over thecourse of tumor growth

We hypothesized that tumor engraftment might transientlyinduce, and then suppress, CXCR3-cognate chemokine produc-tion in the lungs. To explore this possibility, we used a well-characterized model of metastatic-like melanoma and evaluatedCXCL9 and CXCL10 production at various time points followinginjection and establishment of tumors.

In the unperturbed lung compartment, we detected CXCL9 andCXCL10 protein (Fig. 1A and B), consistent with a high level ofcxcl9 and cxcl10 expression in CXCR3-competent C57BL/6 mice(16). Three days after injection of B16 melanoma, we detectedsignificant increases in CXCL9 (Fig. 1A; P < 0.0002) and CXCL10(Fig. 1B;P<0.05) proteins, relative to lungswithout tumor. At thistime point, there was minimal detectable gp100 (melanoma-specific gene) in the lungs (Supplementary Fig. S1A), suggestingminimal residual tumor burden. By 11 days after tumor injection,CXCL9 and CXCL10 production was significantly (P < 0.001 forboth proteins) lower than that observed at day 3. After 18 days oftumor growth, when tumor burden was substantial, chemokine

levels were comparable (CXCL9; P¼ 0.06) or lower (CXCL10; P¼0.0003) than basal levels. From day 3 to 18, CXCL9 and CXCL10were significantly (P < 0.0001 and R2 ¼ 0.7401, and P ¼ 0.0002and R2 ¼ 0.6879, respectively) inversely correlated with gp100signal (Supplementary Fig. S1B and S1C). Cxcl9 and cxcl10expression by qPCR corresponded with observed protein produc-tion (not shown). Thus,melanoma transiently induces the expres-sion and production of CXCL9 and CXCL10 in lungs bearingmetastatic-like tumors.

The observed production of CXCL9 and CXCL10 3 days aftertumor injection is not likely the consequence of tumor cellinjection-induced transient ischemia. We observed no change inCXCL9 and CXCL10 production at day 1 after injection, butsignificant increases in chemokine production by day 2 (Supple-mentary Fig. S2A and S2B). In contrast, Medoff and colleagues

Figure 1.CXCR3-cognate chemokine production is temporally regulated inmelanoma-bearing lungs. Mice were given i.v. B16 injection on day 0. At the time pointsindicated, mice were sacrificed and lungs assessed for CXCL9 protein (A) andCXCL10 protein (B). Expression of Cxcl9 and Cxcl10 correlated with observedprotein profiles (data not shown). In all panels, differenceswere assessed by at test, as indicated: � , P < 0.05; �� , P < 0.01; �� , P < 0.001. ns, not statisticallysignificant.

Clancy-Thompson et al.

Cancer Immunol Res; 3(8) August 2015 Cancer Immunology Research958




(17) reported a rapid and short-lived production of CXCL10 in aninjury-induced ischemia model. These data discount the role oftransient ischemia in chemokine production in our model andsuggest that chemokine production in tumor-burdened lung is aconsequence of tumor engraftment.

To assess whether spontaneous tumors also suppress CXCR3-cognate chemokine production as a function of growth, weassessed the expression of cxcl9 and cxcl10 in early (�5 mm2,day 25) and late (�25 mm2, day 40) Braf/Pten tumors using aninducible model (13). The timing of assessment is offset, relativeto a transplantable model, as inducible tumors require approx-imately 2 weeks to become palpable and assessable (13).

Consistent with our observations in the transplantable model,we observed higher levels of chemokine gene expression in earlytumors, as compared with those in late tumors (SupplementaryFig. S3). Likewise, we have observed a similar temporal regulationof CXCL9 and CXCL10 in subcutaneously implanted B16 mela-noma (data not shown). These data suggest that tumor-inducedCXCR3-cognate chemokine production, and subsequent suppres-sion, may occur in clinically relevant cancers.

Our primary studies evaluated chemokine production in thetumor-bearing lung compartment, rather than in isolated tumors.Thus, our chemokine data from the lung studies represent thetotal tumor-bearing organ. To more precisely define the source of

Figure 2.Cellular sources of CXCL9 and CXCL10in lungs bearing day 3 metastatic-likemelanoma. A–C, light microscopy andimmunohistochemical staining forDAPI, melanoma-specific antigen(TA99), and chemokine (CXCL9-10).Lungs from (A) tumor-free (na€�ve)miceand (B and C) mice i.v. injected 3 daysearlier with B16 melanoma, weresacrificed and assessed for chemokineproduction. D, intracellular flowcytometric evaluation of CXCL9-expressing cells. Lungs from mice withday 3 B16 tumors were processed tosingle-cell suspensions and stained forintracellular CXCL9 and surface lineagemarkers.






chemokine in this model, we assessed CXCL9 and CXCL10 byimmunohistochemical staining of lungs bearing day 3 tumors(Fig. 2A–C), corresponding to peak chemokine production. Spe-cific staining for CXCL9 (Fig. 2B, green) and CXCL10 (Fig. 2C,green) was localized in proximity of melanoma-specific staining(red), including colocalization, suggesting melanoma is asource of chemokine. Melanoma production of chemokine isconsistent with prior observations in patient-derived cell lines(9) and tumors (8), and we observed that B16 melanoma pro-duces CXCL10 in response to IFNg stimulation in vitro (Supple-mentary Fig. S4A). In keeping with this observation, CD45�

CD31�FSChiSSChi cells (putative tumor cells) from early (day3) lungs produced CXCL9, whereas CXCL9 was mostly ablated inthis cell population by day 18, suggesting that melanomamay bea major source of chemokine in this model (Supplementary Fig.S4B). Chemokine production was also observed in non-tumorcells (Supplementary Fig. S5). CD4 T cells and neutrophils are themajor non–tumor CXCL9-producing cell populations in tumor-bearing lungs (Fig. 2D and Supplementary Fig. S5A). Surprisingly,only a small proportion of CD45�CD31þ endothelial cells werepositive for CXCL9 (Fig. 2D). Thus, tumor cells and immune cellsare likely to be the major contributors to CXCR3-cognatechemokine in the lung during early metastatic tumor growth.

Differential IFN production over the course of tumor growthIFNs are major inducers of CXCL9 and CXCL10 expression and

production (18). Therefore, we evaluated IFNg (type II) and IFNa(type I) in our murine tumor model of metastatic-like melanomain the lungs. IFNg production in the lungs was significantly (P <0.005) enhanced at day 3 after tumor induction, relative to micewith no tumor. Importantly, IFNg production was significantlydiminished by day 18, relative to day 3 levels (P< 0.0005) or basallevels (P < 0.01; Fig. 3A). Similarly, Ifna expression was signifi-cantly enhanced on day 3 (P < 0.001) versus mice with no tumor,and significantly reduced from days 3 to 18 (P < 0.0001; Fig. 3B).The induction of IFN expression is not a consequence of transienttumor injection-induced ischemia, as no increase in IFN produc-tion or expression was observed at day 1 after tumor injection(data not shown). Comparable temporal regulation of IFNgexpression was also observed in the Braf/Pten inducible tumormodel (data not shown). These data demonstrate that engraftingmelanomas induce IFNg and IFNa, and these cytokines likelyinduce CXCL9 and CXLC10 production in the tumor-bearingorgan.

Melanoma is an unlikely source of IFN production, and wehave not detected IFNg or IFNa protein or Ifng or Ifna geneexpression in cultured B16or humanmelanoma cell lines, even inresponse to stimulation with exogenous TNFa (data not shown).We assessed IFNg production in nontumor cells by intracellularflow cytometry (9), in tandemwith specific lineage markers. CD4T cells are the predominant nontumor population of IFNg-pro-ducing cells in tumor-bearing lungs (Fig. 3C and SupplementaryFig. S5B); as observed for chemokine, only a small number ofCD45�CD31þ endothelial cells were positive for IFNg .

Differential T-cell infiltration over the course of tumor growthcorresponds with production of CXCR3 ligands

We previously associated survival with the presence of circu-lating CXCR3þCD8þ tumor-specific T cells (10), and the presenceof infiltrating CD8 T cells in human melanoma metastases has

Figure 3.IFN production is temporally regulated in melanoma-bearing lungs. Micewere given i.v. B16 injection on day 0. At the time points indicated, micewere sacrificed and lungs assessed for IFNg protein (A) and Ifnaexpression (B; normalized to GAPDH). Expression of Ifng correlated withobserved protein profiles (data not shown). C, intracellular flowcytometric evaluation of IFNg-expressing cells. Lungs from mice with day3 B16 tumors were processed to single-cell suspensions and stained forintracellular IFNg and surface lineage markers. In A and B, differences wereassessed by a t test, as indicated: � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001. ns, not statistically significant.






Figure 4.CXCR3-expressing Pmel CD8þ T cells traffic to lungs more efficiently than CXCR3-deficient Pmel T cells. To assess T-cell infiltration of tumor-bearing lungs, micewere given i.v. B16 injection on day 0, then received adoptive transfer with either CXCR3þ/þ (Pmel) or CXCR3�/� (Pmel/CXCR3�/�) tumor antigen-specificCD8þ T cells. A, number of recovered Pmel CD8þ T cells from lungs of RAG�/� hosts at various stages of tumor growth. Comparable results were observedusingB6hosts (data not shown). B, number of recovered Pmel CD8þT cells from lungs of RAG�/� and IFNg�/�hosts on day3 after tumor challenge. C, representativeflowcytometry data, gated on live CD45þ cells in the lymphocyte FS/SS gate, from lungs ofmicewith day3 tumors, following adoptive transfer of Pmel (CXCR3wt) orPmel/CXCR3�/� cells. D, number of recovered Pmel or Pmel/CXCR3�/� CD8þ T cells from lungs of mice at day 3 after tumor challenge. E, number of recoveredPmel or Pmel/CXCR3�/� CD8þ T cells from lungs of mice at various times after tumor challenge. In all panels, differences were assessed by t test, asindicated: � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ��, P < 0.0001.






been associated with a defined set of chemokines, includingCXCL9 and CXCL10 (8). Therefore, we hypothesized that T-cellinfiltration of melanoma-engrafted lungs may require CXCR3-cognate chemokines. To assess this possibility, we transferred invitro–activated, tumor-specific (Pmel) CD8þ T cells into unchal-lenged Rag�/� mice or tumor-challenged Rag�/� mice at 3, 11, or18 days after tumor injection, then assessed T-cell infiltration oflungs 1 hour after transfer (14). The majority of activated Pmelcells express CXCR3 (Supplementary Fig. S6).

In agreement with our hypothesis, Pmel CD8þ T cells trafficmore efficiently to metastatic tumor-involved lungs in the earlyphase of growth (day 3), versus lungs bearing more established(day 18) tumors (P < 0.0001; Fig. 4A). T-cell infiltration at day 18was lower (P < 0.001) than basal levels of infiltration in tumor-free mice. Results of these studies were similar in B6 (RagWT)tumor hosts (data not shown). These data demonstrate that T-cellinfiltration of metastatic tumor-bearing lungs correlates withmelanoma-induced production of IFNg and CXCR3-cognate che-mokines, and that T-cell access to the organ compartmentbecomes compromised over the course of tumor growth.

To confirm a requirement for IFNg in the trafficking of trans-ferred Pmel T cells to metastatic melanomas, we used IFNg�/�

mice as tumor hosts. After transfer of Pmel CD8þ T cells intomicewith day 3 inflammatory lung metastases, the number of lung-infiltrating T cells was significantly decreased in IFNg�/� mice, ascompared with comparable tumor-bearing Rag�/� hosts (P <0.001; Fig. 4B) or B6 hosts (P < 0.05; Supplementary Fig. S7A).Interestingly, in the IFNg�/�host, thenumberof transferred T cellsin the spleen was significantly greater than that in tumor-bearinglung (P¼ 0.01; Supplementary Fig. S7B), consistent with the lackof chemoattraction of T cells into inflamed peripheral organs inthese mice. Therefore, T-cell infiltration of metastatic tumor-bearing lungs requires host expression of IFNg , likely as theinducer of CXCL9 and CXCL10.

CXCR3-expressing CD8 T cells are preferentially recruited totumor-bearing lung during early phases of tumor growth

The observed correlation between CXCL9 and CXCL10 pro-duction and T-cell infiltration in lungs suggest that CXCR3 mayplay a significant role in CD8 T-cell access of tumor-bearing lungs.To assess the role of T cell–expressed CXCR3, we compared theinfiltration of in vitro–activated CD8þ Pmel and Pmel/CXCR3�/�

T cells in Rag�/� hosts bearing day 3 lung metastases. Tumorantigen–specific CXCR3þ CD8þ T cells infiltrated tumor-bearinglungs to a greater extent than cells lacking CXCR3 (P < 0.0005; Fig.4C and D), even though adoptively transferred CXCR3þ andCXCR3�/� cells were equivalently represented in the spleen(Supplementary Fig. S7C).

Over the course of tumor growth, infiltration by CXCR3þ PmelT cells was significantly and consistently greater than that ofPmel/CXCR3�/� cells (Fig. 4E); CXCR3þ T-cell infiltration coor-dinated with the level of observed CXCR3 ligands in the organcompartment, whereas infiltration by Pmel/CXCR3�/� T cells didnot significantly changeover the courseof tumorgrowth. Therefore,CXCR3 is critical for T-cell infiltration during early phases of tumordevelopment, but is insufficient to facilitate T-cell access of organswith significant tumor load and a dearth of CXCR3-cognatechemokines.

Consistent with data from Thapa and colleagues (19), weobserved that Pmel/CXCR3�/� CD8 T cells were less capable oflysing target cells (Supplementary Fig. S8A), indicating reduced

effector function. However, pharmacologic activation inducedcomparable proportions of IFNgþ cells from Pmel or Pmel/CXCR3�/� mice (Supplementary Fig. S8B), and ablation ofCXCR3 did not diminish the capacity of the CD8 cells to migratein response to CCR5-specific chemokine (CCL5, SupplementaryFig. S8C). Thus, although loss ofCXCR3maynegatively affect cell-mediated effector function, our short-term studies are designed toassess the impact of CXCR3 on T-cell infiltration (14), and theuncoupling of effector function and migratory activity (19, 20)validates our model for this purpose.

Adenosine signaling suppresses IFN and CXCR3-cognatechemokine production in melanoma

Our data suggest that IFN and CXCR3-cognate chemokinemaybe suppressed in lungs bearing advanced melanomas. However,the mechanism of suppression is undefined. Because free aden-osine accumulates in hypoxic TMEs (21) such as the advancedmetastatic lesions in our model system, and adenosine signalingvia A2 receptors (A2R) suppresses IFN-inducible CXCL9 andCXCL10 production in immune cells (22), we assessed the pos-sibility that adenosine signaling may suppress cytokine andchemokine production in metastasis-bearing lungs. Administra-tion of the clinically available nonselective adenosine receptorantagonist aminophylline (23) to tumor-bearingmice significant-ly increased CXCL9 (P < 0.001), CXCL10 (P < 0.0005), and IFNg(P < 0.01) production in lungs bearing established day 18 mel-anoma metastases (Fig. 5A–C). Aminophylline treatment had noeffect onCXCL9, CXCL10, or IFNg production in tumor-free lungs(Supplementary Fig. S9 and data not shown).

As aminophylline treatment partially restored chemokine pro-duction in lungs with advanced melanoma, we assessed whethertreatment with this adenosine receptor antagonist could enhanceT-cell infiltration in lungs with advanced melanoma that sup-presses CXCR3-cognate chemokine production. Using the adop-tive transfermodel described above, we assessed T-cell infiltrationof lungs bearing day 18 melanoma following treatment withaminophylline or vehicle (PBS). T-cell infiltration was modestlybut significantly (P < 0.05) enhanced by aminophylline treatment(Fig. 6A and B), while T-cell populations in the spleen wereunaffected (Fig. 6C). Importantly, AMO treatment enhanced theantitumor efficacyof adoptively transferredpmel (tumor-specific)T cells into mice with advanced (day 15–18) tumors, as assessedby the level of trp1 gene expression in lungs, whereas adoptivetransfer of pmel T cells intomice without AMOhad no significanteffect on tumor burden (Fig. 6D). Collectively, these data suggestthat suppression of chemokine in tumor-bearing lungs is partiallydue to adenosine accumulation, and that chemotactic activitymay be maintained or restored with specific adenosine receptorantagonists.

Our prior data suggest that melanoma is a source of CXCR3-cognate chemokine in the TME, so we assessed the possibility thatadenosine suppresses tumor production of chemokine. We con-firmed that B16melanoma expresses the adenosine receptor A2AR(ADORA2A; Fig. 7A) as well as ADORA2A and ADORA2B (whichencodes A2BR) gene products (data not shown). In vitro, exoge-nous adenosine suppressedB16productionofCXCL10, even afteractivation of chemokine production via treatment with IFNg (Fig.7B). Importantly, the A2R-specific inhibitor ZM-241,385 (24)significantly reversed adenosine-mediated suppression ofCXCL10 (Fig. 7B). Similarly, five of seven human melanoma celllines tested expressed surface A2AR (representative data for line






VMM12; Fig. 7C), and exogenous adenosine suppressed chemo-kine production by VMM12 melanoma (Fig. 7D).

Although the source of adenosine in the TME remains to bedetermined, we observed an increase in the proportion of regu-

latory (Foxp3þCD4þ) cells in the day 18 TME (SupplementaryFig. S10), and these cells expressed high levels of the ecto-50-nucleotidase CD73, which converts AMP to adenosine. Thus,infiltrating regulatory cells may mediate the accumulation ofbioactive adenosine in the melanoma microenvironment, andthis possibility remains under study. Collectively, these datademonstrate that adenosine can suppress, and adenosine receptorantagonism can restore, chemokineproduction in IFN-stimulatedmelanoma cells.

DiscussionThe failure of immunogenic vaccines to mediate regression of

established tumors or metastases has generally been ascribed toan immunosuppressive TME, which arises as a consequence oftumor-derived immunomodulatory factors and the function ofsuppressor cells. Conversely, vaccines in the adjuvant setting havedemonstratedmodest clinical effects, suggesting that newly form-ing metastases may be more susceptible to T-cell infiltration anderadication. Previous literature has not considered the possibilitythat cytokine and chemokine in the metastatic TME may bedifferentially regulated over the course of tumor growth. In amelanoma metastasis model, we demonstrated a significanttumor-induced production of CXCR3-cognate chemotactic mole-cules in themicroenvironment, but not in systemic circulation. Inanimals with substantial tumor burden, chemokine productionwas suppressed. Thus, our data demonstrate that tumors have atemporal "window of vulnerability" of chemokine productionand associated T-cell infiltration.

In keeping with our previous observation that CXCR3-expres-sing T cells are associated with enhanced survival (10), we foundthat CXCR3 is essential for T-cell infiltration of lungs bearing earlymetastatic lesions. The requirement forCXCR3expressionbyCD8T cells to efficiently infiltrate lungs bearing metastatic tumors,although not previously demonstrated, is consistent with theobserved association between CXCR3 and T-cell access of lungsin situations of viral infection (25, 26), graft rejection (27),chronic inflammation (16, 28), and nonallergic asthma (29).Our data establish the requirement for CXCR3 expression by CD8T cells to efficiently infiltrate melanoma-engrafted lungs.

As vaccination with peptide and adjuvant can induce CXCR3-expressing tumor-specific T cells (30), these data suggest theprotective aspects of adjuvant setting vaccine may arise frompreexisting tumor-reactive CXCR3þCD8þ T cells that rapidlyinfiltrate and eradicate newly arising tumors, whereas establishedtumors may be recalcitrant to infiltration by the same cells. Byextension, these data suggest that tumors may be conditioned forenhanced T-cell infiltration through pharmacologic or immuno-logic interventions to restore chemokine production in the localmicroenvironment, thereby enhancing or inducing the antitumorefficacy of therapeutic-setting vaccines. For example, bacteria havebeen used to induce CXCL9 and CXCL10 in the TME; recentstudies have demonstrated the induction of CXCR3 chemokinesin solid melanomas following infection with Toxoplasma gondii(31) and in lung metastases following infection with Trichinellaspiralis (32).

The rapid expression, then contraction, of IFNg , CXCL9, andCXCL10 in lungs has been observed in an infection model usingchronic Rickettsia conorii infection (33), suggesting that inductionof these factors may be a common response of the reparatorymicroenvironment to tissue damage. Our data extend this

Figure 5.Inflammatory IFN and chemokine levels are increased in aminophylline-treated mice with established lung metastases. Mice were given i.v. B16injection on day 0. Every 3 days, mice were treated with an i.p. injectionof PBS or aminophylline (50 mg/kg). On day 18, mice were sacrificed andproteins were harvested from the lungs. A, CXCL9 production. B, CXCL10production. C, IFNg production. In all panels, differences were assessed by at test: �� , P < 0.01; �� , P < 0.001.






paradigm to cancer, demonstrating that metastatic-like lesions inthe lungs rapidly induce IFNg and chemokine production. Themolecular mechanisms of tumor-induced cytokine and chemo-

kine remain under investigation, although we demonstrate thatmelanomamay be amajor contributor of CXCL9 and CXCL10, inkeeping with our in vitro observation in human melanoma cell

Figure 6.Aminophylline treatment enhances T-cell infiltration of lungs with advanced (day 18) B16 melanoma. Mice were given i.v. B16 injection on day 0. Every3 days, mice were treated with an i.p. injection of PBS or aminophylline (50 mg/kg). Mice received adoptive transfer of 1 � 106 Pmel CD8þ T cells on day 18(for tracking studies A–C) or day 15 (for tumor control study, D). A, representative flow cytometry of Thy1.1þCD8þ Pmel T cells in mice treated with PBS oraminophylline. B, number of recovered Pmel CD8þ T cells from lungs of B6 mice treated with PBS or aminophylline. C, number of recovered Pmel CD8þ T cellsfrom spleens of B6 mice treated with PBS or aminophylline. D, tumor burden in lungs of mice treated with PBS or aminophylline, without or with adoptivetransfer (day 15) of Pmel T cells. Assessment of tumor burden at day 18. Differenceswere assessed by a t test: � , P <0.05; �� , P <0.01; �� , P <0.001; ns, not statisticallysignificant.






lines (9). Surprisingly, a significant number of IFNg- and chemo-kine-producing CD4 T cells, but notNK cells, are apparent in earlymelanoma-infiltrated lungs. The role of these CD4 cells, and themechanism by which they infiltrate the lungs, remains underinvestigation.

The observation that melanoma suppresses IFNs, CXCL9, andCXCL10 over the course of metastatic tumor growth has not beenreported, and these data have implications for T cell–mediatedtherapy of cancer. To explain the active suppression of IFNs andchemokines, as opposed to a failure to induce these factors, weassessed the activity of the adenosine signaling axis in this model.Present in both intra- and extracellular compartments, in thenanomolar range (34), adenosine suppresses antitumorimmune responses and can enhance tumor outgrowth. Cekic andcolleagues (15) demonstrated that antagonism of adenosine

slows the progression of bladder or breast carcinoma growing assolid tumors in a CXCR3-dependent manner; therefore, we eval-uated the possibility that adenosine signaling blockade, in thecontext of metastatic melanoma in the lung, may influence theCXCR3 chemotactic axis. We demonstrate that interfering withadenosine signaling restores IFNg , CXCL9, andCXCL10 levels andpartially reconstitutes T-cell infiltration in situ. CD4 cells aresignificant sources of IFNg in our model, and adenosine has beenshown to suppress IFNg production in CD4 cells (35). Further-more, we found that adenosine restores IFNg-induced productionof chemokines by melanoma. Therefore, we conjecture thathypoxia in the microenvironment of advanced metastatic diseaseleads to an accumulation of adenosine, which suppresses IFNgand CXCR3-cognate chemokine production, and we are currentlyvalidating these mechanisms. Interestingly, the capacity of

Figure 7.Adenosine suppresses, and adenosinereceptor 2 antagonist restores, CXCL10production by melanoma cells in vitro.A, flow cytometric staining of B16melanoma for adenosine receptorA2A (ADORA2A). Shaded histogram,unstained; open histogram, A2AR-specific staining. B, adenosine regulatesCXCL10 production by B16 melanoma.Supernatant from B16 melanoma,cultured in the presence or absence ofexogenous IFNg (0 pg, 2,500 pg), ADO(0 mmol/L, 500 mmol/L, 1,000 mmol/L),and ZM241,385 (ZM; 0 mmol/L,10 mmol/L), was assessed for CXCL10production by specific ELISA. C, flowcytometric staining of human VMM12melanoma for adenosine receptor A2A

(ADORA2A). Shaded histogram,unstained; open histogram, A2AR-specific staining. D, adenosine regulatesCXCL10 production by human VMM12melanoma. Supernatant from VMM12melanoma, cultured in the presence orabsence of exogenous IFNg (0 pg,2,500 pg) and adenosine (0 mmol/L,500 mmol/L) was assessed for CXCL10production by specific ELISA.Differences were assessed by a t test:� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






adenosine to directly suppress CXCL10 production in IFNg-stim-ulated melanoma cells indicates a specific blockade of geneexpression, suggesting that adenosine-mediated suppression ofchemokine in the TME may occur even in the presence of exog-enous IFNs. Our data also demonstrate that adenosine receptor–mediated suppression of CXCR3-cognate chemokine extends tohuman melanoma cells, consistent with the single prior report ofA2AR in a single human melanoma cell line (36). Our datademonstrate that blocking adenosine A2 receptor function issufficient to restore chemotactic gradients, and our ongoingstudies will elaborate the relative contributions of A2AR andA2BR.

A recent study has demonstrated that cutaneous melanomasdecrease CXCL9 production because of immune editing andselection of CXCL9-deficient clones (37). We have not excludedthe possibility of immune editing in the metastatic-like model;however, themaintenance of Ifng,Cxcl9, andCxcl10 expression inlate-stage metastases following aminophylline treatment sup-ports an argument against the selection and predominance ofloss-of-function clones in our model.

The paucity of responses to immunogenic vaccines and adop-tive transfer protocols suggests the need for adjuvant therapy toinduce or restore T-cell infiltration into tumor compartments.Although chemokine expression is not sufficient to induce orrestore antitumor function, chemokines are essential for effectiveT-cell chemoattraction and therefore an important aspect ofcancer immunotherapy. Collectively, our data demonstrate thatCXCR3-cognate ligand expression is required for efficient T-cellaccess of tumor-infiltrated lungs, but these ligands are expressedin a temporally restricted pattern. We further demonstrate ameans to partially overcome tumor-induced suppression of lym-phocyte chemotaxis, suggesting that metastases may be condi-tioned for enhanced or restored chemokine expression and T-cellinfiltration, thereby potentially enhancing the therapeutic efficacyof immunogenic but clinically ineffective vaccine platforms.

Disclosure of Potential Conflicts of InterestD.W.Mullins is Chief ScientificOfficer atQuBiologics and reports receiving a

commercial research grant from the same. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: E. Clancy-Thompson, M.J. Turk, D.W. MullinsDevelopment of methodology: E. Clancy-Thompson, T.J. Perekslis, W. Cro-teau, Y.H. Huang, D.W. MullinsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Clancy-Thompson, W. Croteau, M.P. Alexander,T.B. Chabanet, Y.H. Huang, D.W. MullinsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Clancy-Thompson, T.B. Chabanet, Y.H. Huang,D.W. MullinsWriting, review, and/or revision of themanuscript: E. Clancy-Thompson, Y.H.Huang, D.W. MullinsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Clancy-Thompson, T.B. Chabanet, D.W.MullinsStudy supervision: D.W. Mullins

AcknowledgmentsLenoraD. Nunnley provided technical support for some studies. The authors

thank John DeLong and Dr. Jacqueline Channon-Smith (DartLab, the GeiselSchool of Medicine's Immune Monitoring and Cytometry Core Facility) forexpert assistance with flow cytometry studies, and Jennifer Fields andDr. StevenN. Fiering (Dartmouth Transgenic and Genetic Construct Shared Resource) forexpert assistance with mouse models. Maryann Mikucki and Dr. Sharon Evans(Department of Immunology, Roswell Park Cancer Institute) provided insight-ful comments and suggestions.

Grant SupportSupport for these studies was provided, in part, by the Geisel School of

Medicine at Dartmouth's Immunology Training Grant (USPHS T32 AI007363to Dr. Charles L. Sentman, sub-award to E. Clancy-Thompson); the DartmouthCenter for Molecular, Cellular, and Translational Immunology (USPHS P30GM103415 toDr.WilliamR.Green, sub-award toD.W.Mullins); the JoannaM.NicolayMelanomaResearch Foundation (Research Scholar Award, to E.Clancy-Thompson); theMelanoma Research Alliance (Young Investigator Award, to D.W. Mullins); and the USPHS (R01 AI089805, to Y.H. Huang; and R01CA134799, to D.W. Mullins).

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 January 14, 2015; revised May 16, 2015; accepted May 27, 2015;published OnlineFirst June 5, 2015.

References1. Slingluff CL, Petroni GR, YamshchikovGV, BarndDL, Eastham S,Galavotti

H, et al. Clinical and immunologic results of a randomized phase II trial ofvaccination using four melanoma peptides either administered in granu-locyte-macrophage colony-stimulating factor in adjuvant or pulsed ondendritic cells. J Clin Oncol 2003;21:4016–26.

2. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: movingbeyond current vaccines. Nat Med 2004;10:909–15.

3. Slingluff CL, Yamshchikov G, Neese P, Galavotti H, Eastham S, EngelhardVH, et al. Phase I trial of amelanoma vaccinewith gp100(280-288) peptideand tetanus helper peptide in adjuvant: immunologic and clinical out-comes. Clin Cancer Res 2001;7:3012–24.

4. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strat-egies that aremediatedby tumor cells. AnnuRev Immunol 2007;25:267–96.

5. Clemente CG, Mihm MC, Bufalino R, Zurrida S, Collini P, Cascinelli N.Prognostic value of tumor infiltrating lymphocytes in the vertical growthphase of primary cutaneous melanoma. Cancer 1996;77:1303–10.

6. Erdag G, Schaefer JT, Smolkin ME, Deacon DH, Shea SM, Dengel LT, et al.Immunotype and immunohistologic characteristics of tumor-infiltratingimmune cells are associated with clinical outcome in metastatic melano-ma. Cancer Res 2012;72:1070–80.

7. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pag�esC, et al. Type, density, and location of immune cells within humancolorectal tumors predict clinical outcome. Science 2006;313:1960–4.

8. Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, et al.Chemokine expression in melanoma metastases associated with CD8þ T-cell recruitment. Cancer Res 2009;69:3077–85.

9. Dengel LT, Norrod AG, Gregory BL, Clancy-Thompson E, Burdick MD,Strieter RM, et al. Interferons induce CXCR3-cognate chemokine produc-tion by human metastatic melanoma. J Immunother 2010;33:965–74.

10. Mullins IM, Slingluff CL, Lee JK, Garbee CF, Shu J, Anderson SG, et al. CXCchemokine receptor 3 expression by activated CD8þ T cells is associatedwith survival in melanoma patients with stage III disease. Cancer Res2004;64:7697–701.

11. Hsueh EC, Essner R, Foshag LJ, Ollila DW, Gammon G, O'Day SJ, et al.Prolonged survival after complete resection of disseminated melanomaand active immunotherapy with a therapeutic cancer vaccine. J Clin Oncol2002;20:4549–54.

12. Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky WE,et al. Braf(V600E) cooperates with Pten loss to induce metastatic mela-noma. Nat Genet 2009;41:544–52.






13. Steinberg SM, Zhang P, Malik BT, Boni A, Shabaneh TB, Byrne KT, et al.BRAF inhibition alleviates immune suppression inmurine autochthonousmelanoma. Cancer Immunol Res 2014;2:1044–50.

14. Fisher DT, ChenQ, Skitzki JJ, Muhitch JB, Zhou L, Appenheimer MM, et al.IL-6 trans-signaling licenses mouse and human tumor microvascular gate-ways for trafficking of cytotoxic T cells. J Clin Invest 2011;121:3846–59.

15. Cekic C, Sag D, Li Y, Theodorescu D, Strieter RM, Linden J. Adenosine A2Breceptor blockade slows growth of bladder and breast tumors. J Immunol2012;188:198–205.

16. Nie L, Xiang R, Zhou W, Lu B, Cheng D, Gao J. Attenuation of acute lunginflammation induced by cigarette smoke inCXCR3 knockoutmice. RespirRes 2008;9:82.

17. Medoff BD, Wain JC, Seung E, Jackobek R, Means TK, Ginns LC, et al.CXCR3 and its ligands in a murine model of obliterative bronchiolitis:regulation and function. J Immunol 2006;176:7087–95.

18. Groom JR, Luster AD. CXCR3 ligands: redundant, collaborative andantagonistic functions. Immunol Cell Biol 2011;89:207–15.

19. Thapa M, Carr DJJ. CXCR3 deficiency increases susceptibility to genitalherpes simplex virus type 2 infection: uncoupling of CD8þ T-cell effectorfunction but not migration. J Virol 2009;83:9486–501.

20. Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res 2011;317:620–31.

21. Stagg J, Smyth MJ. Extracellular adenosine triphosphate and adenosine incancer. Oncogene 2010;29:5346–58.

22. Wallace KL,Marshall MA, Ramos SI, Lannigan JA, Field JJ, Strieter RM, et al.NKT cells mediate pulmonary inflammation and dysfunction in murinesickle cell disease through production of IFN-gamma and CXCR3 chemo-kines. Blood 2009;114:667–76.

23. Hochwald C, Kennedy K, Chang J, Moya F. A randomized, controlled,double-blind trial comparing two loading doses of aminophylline.J Perinatol 2002;134:275–8.

24. Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PW, Jones G, et al.The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2aselective adenosine receptor antagonist. Br J Pharmacol 1995;115:1096–102.

25. Kohlmeier JE, Cookenham T, Miller SC, Roberts AD, Christensen JP,Thomsen AR, et al. CXCR3 directs antigen-specific effector CD4þ T cellmigration to the lung during parainfluenza virus infection. J Immunol2009;183:4378–84.

26. Lindell DM, Lane TE, Lukacs NW. CXCL10/CXCR3-mediated responsespromote immunity to respiratory syncytial virus infection by augment-

ing dendritic cell and CD8(þ) T cell efficacy. Eur J Immunol2008;38:2168–79.

27. Seung E, Cho JL, Sparwasser T, Medoff BD, Luster AD. Inhibiting CXCR3-dependent CD8þ T cell trafficking enhances tolerance induction in amouse model of lung rejection. J Immunol 2011;186:6830–8.

28. Lin Y, Yan H, Xiao Y, Piao H, Xiang R, Jiang L, et al. Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knock-out mice. Respir Res 2011;12:123.

29. Barbarroja-Escudero J, Prieto-Martin A, Monserrat-Sanz J, Reyes-Martin E,Diaz-Martin D, Antolin-Amerigo D, et al. Abnormal chemokine receptorprofile on circulating T lymphocytes from nonallergic asthma patients. IntArch Allergy Immunol 2014;164:228–36.

30. Clancy-Thompson E, King LK, Nunnley LD, Mullins IM, Slingluff CL,Mullins DW. Peptide vaccination in montanide adjuvant induces andGM-CSF increases CXCR3 and cutaneous lymphocyte antigen expres-sion by tumor antigen-specific CD8 T cells. Cancer Immunol Res2013;1:332–9.

31. Baird JR, Byrne KT, Lizotte PH, Toraya-Brown S, Scarlett UK, AlexanderMP,et al. Immune-mediated regression of established B16F10 melanoma byintratumoral injection of attenuated toxoplasma gondii protects againstrechallenge. J Immunol 2013;190:469–78.

32. Kang Y-J, Jo J-O, Cho M-K, Yu H-S, Leem S-H, Song KS, et al. Trichinellaspiralis infection reduces tumor growth and metastasis of B16-F10 mel-anoma cells. Vet Parasitol 2013;196:106–13.

33. Valbuena G, Bradford W, Walker DH. Expression analysis of the T-cell-targeting chemokines CXCL9 and CXCL10 in mice and humans withendothelial infections caused by rickettsiae of the spotted fever group.Am J Pathol 2003;163:1357–69.

34. Fredholm BB. Adenosine, an endogenous distress signal, modulates tissuedamage and repair. Cell Death Differ 2007;14:1315–23.

35. Erdmann AA, Gao Z-G, Jung U, Foley J, Borenstein T, Jacobson KA, et al.Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo. Blood 2005;105:4707–14.

36. Merighi S, Mirandola P, Milani D, Varani K, Gessi S, Klotz K-N, et al.Adenosine receptors as mediators of both cell proliferation and celldeath of cultured human melanoma cells. J Invest Dermatol 2002;119:923–33.

37. Petro M, Kish D, Guryanova OA, Ilyinskaya G, Kondratova A, Fairchild RL,et al. Cutaneous tumors cease CXCL9/Mig production as a result of IFN-g-mediated immunoediting. J Immunol 2013;190:832–41.






2015;3:956-967. Published OnlineFirst June 5, 2015.Cancer Immunol Res Eleanor Clancy-Thompson, Thomas J. Perekslis, Walburga Croteau, et al. Metastatic-like DiseaseChemokine Production and T-cell Infiltration of Lungs Bearing Melanoma Induces, and Adenosine Suppresses, CXCR3-Cognate

Updated version

10.1158/2326-6066.CIR-15-0015doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/06/04/2326-6066.CIR-15-0015.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/3/8/956.full#ref-list-1

This article cites 37 articles, 18 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/3/8/956.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerimmunolres.aacrjournals.org/content/3/8/956To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-15-0015

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/06/04/2326-6066.CIR-15-0015.DC1

http://cancerimmunolres.aacrjournals.org/content/3/8/956.full#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/3/8/956.full#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/3/8/956


Melanoma Induces, and Adenosine Suppresses, CXCR3 ... · Eleanor Clancy-Thompson1,2,Thomas J....

Documents

Transcript of Melanoma Induces, and Adenosine Suppresses, CXCR3 ... · Eleanor Clancy-Thompson1,2,Thomas J....