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Research Article

CD137 (4-1BB) Costimulation Modifies DNAMethylation in CD8þ T Cell–Relevant GenesM. Angela Aznar1, Sara Labiano1, Angel Diaz-Lagares2,3, Carmen Molina1,Saray Garasa1, Arantza Azpilikueta1, I~naki Etxeberria1, Alfonso R. Sanchez-Paulete1,Alan J. Korman4, Manel Esteller2,3,5,6, Juan Sandoval7, and Ignacio Melero1,3

Abstract

CD137 (4-1BB) costimulation imprints long-term changes thatinstruct the ultimate behavior of T cells that have previouslyexperienced CD137 ligation. Epigenetic changes could providea suitable mechanism for these long-term consequences.Genome-wide DNA methylation arrays were carried out onhuman peripheral blood CD8þ T lymphocytes stimulatedwith agonist monoclonal antibody to CD137, including ure-lumab, which is in phase I/II clinical trials for cancer immu-notherapy. Several genes showed consistent methylation pat-terns in response to CD137 costimulation, which were con-firmed by pyrosequencing in a series of healthy donors. CD96,HHLA2, CCR5, CXCR5, and CCL5 were among the immune-related genes regulated by differential DNA methylation,

leading to changes in mRNA and protein expression. Thesegenes are also differentially methylated in na€�ve versusantigen-experienced CD8þ T cells. The transcription factorTCF1 and the microRNA miR-21 were regulated by DNAmethylation upon CD137 costimulation. Such gene-expres-sion regulatory factors can, in turn, broaden the effects ofDNA methylation by controlling expression of their targetgenes. Overall, chromatin remodeling is postulated to leaveCD137-costimulated T lymphocytes poised to differentiallyrespond upon subsequent antigen recognition. Accordingly,CD137 connects costimulation during priming to genome-wide DNA methylation and chromatin reprogramming.Cancer Immunol Res; 6(1); 69–78. �2017 AACR.

IntroductionCostimulation dictates the outcome of antigen recognition

by T cells. Immediate changes in signaling, gene expression,and metabolism take place if costimulation is provided (1)but, in addition, subtle long-term changes occur that leaveT cells poised to more robustly respond if challenged at alater time point with antigen (2). Several gene-expression

control mechanisms may be involved in such long-term reg-ulation. Transcription factors do not adequately explain thelong-term effects, which suggests a role for epigenetic mod-ifications (2, 3). Epigenetic control of gene expression acts as aswitch to either induce or repress the transcriptional activityof multiple genes implicated in different physiological andpathological conditions. Specific epigenetic mechanisms havebeen identified as being responsible for regulating the expres-sion of certain immune-related genes (3, 4). One of theseepigenetic mechanisms is DNA methylation, which consists ofthe addition of a methyl group to the 50 carbon of cytosinewithin cytosine-guanine dinucleotides (CpG). CpG DNAmethylation is considered perhaps the most fundamentalmolecular phenomenon determining chromatin accessibilityto the transcriptional machinery and thus leads to gene expres-sion regulation. Gene-specific DNA methylation is largelydependent on the activity of DNA methyltransferases (DNMT1,DNMT3a, and DNMT3b) that catalyze the transfer of a methylgroup from S-adenosyl methionine to DNA (5). These enzymesare drawn onto selective genome locations to methylate cyto-sine bases in a sequence-specific fashion by poorly understoodtargeting mechanisms. Such methylation patterns are subse-quently inherited by daughter cells following mitoses (6).Alterations in methylation patterns influence the balance oftranscripts in cells and contribute to pathological conditionssuch as cancer and the deregulation of the immune system (7).

Critical loci in T lymphocytes are regulated by genemethylationand chromatin accessibility, including the FOXP3 locus in naturalTregs (8), the PD-1 locus (PDCD1) in exhausted T cells (9), andthe differentiation to the IFNg-producing phenotype under theinfluence of IL12 (10). How the epigenetic machinery selectivelycontrols these phenomena in a gene-specific manner remains

1Center for Applied Medical Research (CIMA), University of Navarra, Pamplona,Spain. 2Cancer Epigenetics and Biology Program (PEBC), Bellvitge BiomedicalResearch Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Catalonia,Spain. 3CIBERONC—Centro virtual de Investigacion Biomedica en red de Onco-logia, Madrid, Spain. 4Bristol-Myers Squibb, Redwood City, California. 5Departa-ment de Ci�encies Fisiol�ogiques II, Escola de Medicina, Universitat de Barcelona,Barcelona, Catalonia, Spain. 6Instituci�o Catalana de Recerca i Estudis Avancats(ICREA), Barcelona, Catalonia, Spain. 7Biomarkers and Precision Medicine Unit(UByMP), Epigenomics Core Facility, Instituto Investigaci�on Sanitaria La Fe(IISlaFe), Valencia, Spain.

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

M.A. Aznar, S. Labiano, and A. Diaz-Lagares contributed equally to this article.

J. Sandoval and I. Melero share senior authorship of this article.

Current address for A. Diaz-Lagares: Translational Medical Oncology (Onco-met), Roche-CHUS Joint Unit, Health Research Institute of Santiago (IDIS),University Clinical Hospital of Santiago (CHUS), 15706 Santiago de Compost-able, Galicia, Spain.

Corresponding Author: Ignacio Melero, CIMA and Clinica Universidad deNavarra. Avenida Pio XII, 55. 31008 Pamplona, Spain. Phone 34-948194700;Fax: 34-948194717; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0159

�2017 American Association for Cancer Research.

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poorly understood. Genome-wide approaches to resolving thisissue have seldom been undertaken (3, 9).

In T-cell activation, costimulation via the TNFR family mem-bers is key to survival, acquisition of effector functions, andmemory differentiation (11–13). CD137 (4-1BB, TNFRSF9) isnot an exception (14). It gains surface expression on T cells onlyfollowing TCR-mediated priming (15), although its levels areaugmented by CD28 costimulation (16). Ample experimentalevidence shows that when CD137 meets its ligand or agonistmonoclonal antibodies (mAb) onCD8þ T cells, effector functions(17), survival (18), and memory generation are costimulated(13, 19–21). In this sense, agonist mAbs potentiate curativeimmune responses in mice bearing tumors through immunemechanisms mediated primarily by CD8þ cytotoxic T lympho-cytes (14, 22). In this regard, the fully human agonist mAbs toCD137, urelumab (23) and utomilumab (24), are undergoingclinical trials as single agents or in combination with otherimmunostimulatory mAbs (14). In rodent models, CD137engagement leads to more robust T-cell responses, even whensuch mice are rechallenged with cognate antigen months aftertreatment (25, 26), indicating the need for long-term regulationmechanisms imprinted during the primary response (14).

Hypothesizing that such long-term effects of CD137 ligationcould be the consequence of epigenetic changes encompassingchromatin remodeling, we performed experiments with genome-wide high-throughput DNA methylation arrays to identifyimmune genes on which CD137 would influence cytosine meth-ylation patterns at specific motifs. Using human primary CD8þ Tcells from series of healthy volunteers, the DNA methylationchanges were confirmed and found to result in up- or down-regulation of mRNA and protein expression of such genes.

Materials and MethodsT-cell isolation and T-cell culture

HumanCD8þ T lymphocytes were isolated from the peripheralblood of healthy donors by Ficoll gradients, following a negativeselection with CD8þ T Cell Isolation Kit by autoMACS Pro(Miltenyi Biotec). Blood samples were obtained from NavarraBlood and Tissue Bank. Navarrabiomed Biobank, Navarra HealthDepartment. CD8þ T lymphocytes were activated in 12-wellplates previously coated with anti-CD3e (1 mg/mL, clone OKT3)and anti-CD137 (10 mg/mL, 6B4 or urelumab) or respectiveisotype-matched control Ab (10 mg/mL) at 1.75 � 106 cells/wellin RPMI 1640 medium (Gibco) supplemented with 10% FBS(Sigma-Aldrich), 100 IU/mL penicillin and 100 mg/mL strepto-mycin (Gibco) for 5 days (activation period). On day 5, Tlymphocyteswere transferredonto12-well plates in culturemediasupplemented with hIL7 (25 ng/mL, Immunotools) for another 5days (resting period). Restimulation was attained by transferringof T cells to plates coated with anti-CD3e mAb (1 mg/mL, cloneOKT3).

DNA extraction and genome-wide DNA methylation arraysDNA from CD8þ T lymphocytes was isolated using DNeasy

Blood& Tissue kit (Sigma) and quantified byQuant-iT PicoGreendsDNAReagent (Invitrogen). The integritywas analyzed in a 1.3%agarose gel. Bisulfite conversion of 600 ng of each DNA samplewas performed according to the manufacturer's recommendationfor Illumina Infinium Assay. Effective bisulfite conversion waschecked for three controls that were converted simultaneously

with the samples. Four microliters of bisulfite converted DNAwere used to hybridize on Infinium Human Methylation 450BeadChip, following the Illumina Infinium HD Methylationprotocol. Chip analysis was performed using Illumina HiScanSQ fluorescent scanner. The intensities of the images wereextracted using GenomeStudio (2010.3) Methylation module(1.8.5) software. The methylation score of each CpG is repre-sented as the beta (b) value.

The 450KDNAmethylation array by Illumina is an established,highly reproducible method for DNA methylation detection andhas been validated in two independent laboratories (27). The450KDNAmethylation array includes 485,764 cytosine positionsof the human genome thatwerefiltered by sex chromosomeCpGs(avoiding sex link alterations) and nonvalid CpGs (P < 0.001).The intensities of the images were extracted and normalized usingGenomeStudio (2011.1) Methylation module (1.9.0) software.

For determining differentially methylated CpGs an analysisusing an absolute difference in beta values of 0.25 and a standarddeviation <0.1were used for selecting themost relevant positions.

PyrosequencingPyrosequencing analyses to determine CpGmethylation status

were developed as previously described (28). Briefly, a minimumof 500 ng of DNA were converted using the EZ DNAmethylationGold (ZYMO RESEARCH) bisulfite conversion kit following themanufacturer's recommendations. Specific sets of primers for PCRamplification and sequencing were designed using specific soft-ware (PyroMark assay design version 2.0.01.15). Primersequences were designed, when possible, to hybridize withCpG-free sites to ensure methylation-independent amplification(see Supplementary Table S3). PCR was performed under stan-dard conditions with biotinylated primers and the PyroMarkVacuum Prep Tool (Biotage) was used to prepare single-strandedPCR products according to the manufacturer's instructions. PCRproducts were observed on 2% agarose gels before pyrosequen-cing. Reactions were performed in a PyroMark Q24 Systemversion 2.0.6 (Qiagen) using appropriate reagents and protocols,and the methylation value was obtained from the average of theCpG dinucleotides included in the sequence analyzed. Controlsto assess correct bisulfite conversion of the DNA were included ineach run, as well as sequencing controls to ensure the reliability ofthe measurements. Graphic representation of methylation valuesshows bars identifying CpG sites that present percentage meth-ylation values.

RNA extraction and qRT-PCRT-lymphocyte samples were collected at activation, resting

phase, and restimulation time points. Total RNA extraction wascarried out by using TRIzol (Invitrogen) following reverse tran-scriptions with M-MLV reverse transcriptase (Invitrogen). Quan-titative RT-PCR (qRT-PCR) was performed with iQ SYBR greensupermix in a CFX real-time PCR detection system (Biorad).Primer pairs used to detect gene expression are shown in Sup-plementary Table S3. Mature miR-21 expression was assessed byreal-time PCR analysis using Taqman microRNA assays (LifeTechnologies). Reverse transcription was performed on RNAusing the TaqMan MicroRNA Reverse Transcription Kit (cat no4366596). Quantitative RT-PCR was performed with 2� TaqmanFast Universal PCRMaster Mix (cat no 4366072) according to themanufacturer's protocol, with specific TaqMan primers. Gene and

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(MIR21) expression data were normalized with levels of thehousekeeping geneH3 and RNU6B, respectively, and representedaccording to this formula 2DCt (CtH3 or RNU6 - Ct gene), where Ctcorresponds to cycle number.

Flow cytometry and ELISAT lymphocytes were prestained with the Zombi NIR Fixable

viability kit (Biolegend) as a live/deadmarker and pretreatedwithBeriglobin before staining. Surface staining was performed withthe following mAbs purchased from Biolegend: CD8-BV510(5K1), CD96-PE (NK92.39), CCR5-PerCPC5.5 (HEK/1/85a),CXCR5-BV421 (J252D4), mouse IgG1-PE and IgG1-BV421(MOPC-21), and rat IgG2a-PerCPC5.5 (RTK2758) as anisotype-matched negative control. The True-Nuclear Transcrip-tion Factor Buffer Set (Biolegend) was used for intracellularstaining of TCF1 using anti–TCF1-AF647 (7F11A10) and mouseIgG1-AF647 (MOPC-21), both purchased from Biolegend. Cellacquisition was carried out with FACSCanto II and FlowJo (Trees-tar) software was used for data analysis.

CCL5 protein expression was measured from the supernatantsof CD8þ T cell cultures using the RANTES (CCL5) human Simple-Step ELISA Kit (Abcam, ref AB174446).

Staining and culture of FACS sortedna€�ve,memory, and effectorCD8þ T-cell subsets

Human CD8þ T lymphocytes were immunomagnetically puri-fied from peripheral blood of healthy donors as described inMaterial and Methods. Subsequently, na€�ve and antigen-experi-enced T-cell subsets were FACS-sorted as previously described(29). Briefly, CD8þ T cells were pretreated with Beriglobin beforestaining with the following antibodies for cell surface markers:CD8-APC (RPA-T8), CD27-FITC (o323), CD62-L PE (DREG-56),and CD45RA-PerCPC5.5 (HI100) and sorted in a FACSAria (BD).Each purified subpopulation was stained with either Cell TraceViolet (Invitrogen) or CFSE (BD) or left unstained and followingprelabeling populations were remixed. Different combinations ofthese stainings were performed to have cultures with each pop-ulation stained with both cell dyes, rendering similar results. Theresulting remixed CD8þ T cells were activated with plate coatedanti-CD3 mAb and costimulatory mAb (Fig. 1) in 96-well platesin identical density (cells/cm2) for 5 days. An identical experi-mental procedure described in theMaterials andMethods sectionand depicted in Fig. 1 were carried out.

Dendritic cell differentiation and chemotaxis assaysCD14 cells from PBLs of healthy donors were purified with

CD14 MicroBeads (Miltenyi) and cultured in AIM V Medium(Gibco) with 1,000 u/mL of rhu-IL4 (R&D) and huGM-CSF(Bayer). After 6 days, cells were collected, washed, and seeded in8-mmpore polycarbonate Transwell (Costar), at 105 cells per well.

The percent migration of dendritic cells (DC) to CCR5was assessed toward recombinant human CCL5 (Preprotech;300ng/mL) and toward supernatants derived from the 36-hour–restimulated CD8þ T cells that had been previously acti-vated in presence of either urelumab or hIgG (from Fig. 4B).Following a 12-hour culture in the Transwell setting,migratedDCof the bottom chambers were recovered and counted. The per-centage of migration computes is referred to input DC. To blockCCL5-driven cell migration, DCs were incubated for 1 hour with0.8 mg/mL of human CCL5-blocking antibody (R&D) before themigration assay. All the experiments were performed in triplicate.

Statistical analysisPrism software (GraphPad Software, Inc.) was used to analyze

statistical differences of absolute methylation level and mRNAand protein expression of target genes by applying the pairedStudent t test, Mann–Whitney U test or the Wilcoxon paired test.Values of �, P < 0.05; ��, P < 0.01; ��, P < 0.001 were consideredsignificant.

ResultsAgonist CD137mAb regulates theDNAmethylation of relevantCD8þ T-cell genes

Our initial hypothesis was that CD137 signaling in primed Tlymphocytes would result in chromatin remodeling throughsequence-specific genomic DNAmethylation. To study modifica-tions in DNA methylation at the genomic level, we used cultureplate-bound CD3e mAb to mimic TCR priming together withurelumab (23) or 6B4 (30) as CD137 agonists (Fig. 1A). CD8þ Tcells were immunomagnetically isolated from the blood ofhealthy donor volunteers and seeded onto plates coated withanti-CD3e and anti-CD137. At the indicated points of time,samples were retrieved to isolate genomic DNA, mRNA, or tomeasure protein expression in the cells and supernatants. In theprocess of stimulation in culture, samples were transferred on dayþ5 to antibody-free plates to resemble a return-back to a restingstatus in the presence of the homeostatic cytokine IL7. On dayþ10 of culture, T cells were stimulated again with solid-phasebound anti-CD3e as a surrogate of a subsequent antigen encoun-ter. Samples were collected at a series of time points following thissecondary stimulation. This experimental approach sought toobserve changes caused by costimulation that would becomeimprinted into the chromatin of CD137-costimulated CD8þ Tcells. In order to assess nuclear gene DNA methylation in agenome-wide fashion, the 450KDNA-methylation arraywas usedin a series of CD8þ T cells from three independent individualscostimulatedbyurelumab (or control IgG4mAb)orby6B4actingon CD8þ T cells from an additional unrelated donor. Differentialmethylation analyses were performed at day þ10, representing Tlymphocytes in which the CD137-costimulation epigenetic mod-ifications would be already established, potentially leaving thecells poised to long-term respond in a distinct manner.

At day þ10 of CD8þ T-cell culture, 1,028 differentially meth-ylated CpGs corresponding to 907 genes (Fig. 1B) were found tobe modified by CD137 costimulation in their methylation statusat specified CpGs, as determined by an unbiased bioinformaticanalyses at consistent loci either after urelumab or 6B4 activation(Supplementary Tables S1 and S2, respectively). As a generaltendency, most of the changes in CpGs whose methylation statuswas modified in response to anti-CD137 mAb consisted ofdemethylation (87% CpGs for urelumab; 69% CpGs for 6B4)rather than hypermethylation. From the list of 52 genes that weredifferentially methylated by CD137 costimulation observedboth with urelumab and 6B4 (Fig. 1B), several genes (in bold)attracted our attention for their well-described involvement inimmune cell performance. This hand-picked listwas supported byPubMed publication searches andGeneOntology (GO) analyses,that showed an enrichment of immune response functions(GO:0006955, FDR¼ 0.0129). This group includes genes relatedto T-cell costimulation/coinhibition, inflammation and inflam-matory chemotaxis, immune cell transcription factors, and themicroRNA miR-21.

CD137 Costimulation and Epigenetic Reprogramming

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To confirm the reproducibility of these findings, we ana-lyzed the identified methylated or demethylated immune-relevant sequences by pyrosequencing in a series of unrelatedindividuals (n ¼ 19). As we hypothesized, these genes weremodified in a consistent fashion with the findings reported bythe DNA-methylation microarrays, following both urelumab-or 6B4-elicited costimulation (Fig. 2A and B).

DNA-methylation patterns are known to be different in na€�veversus antigen-experienced CD8þ T lymphocytes from humanperipheral blood. DNA-methylation status of TCF7 and CCL5loci, whichwere identified in our genome-wide screenings (Fig. 1)as differentially methylated following CD137 ligation, is indeedknown to be differentially methylated in na€�ve versus memoryCD8þ T cells (4, 31). In fact, when we FACS-sorted na€�ve versusmemory and effector CD8þ T cells from peripheral blood (Sup-plementary Fig. S1A), we saw that our most relevant identifiedgenes followed similar DNA methylation pattern in peripheralblood na€�ve versus memory CD8þ cells (CD96, HHLA2, MIR21,and CXCR5; Supplementary Fig. S1B).

To address whether the frequencies of originally na€�ve versusmemory cells changed in the resulting cultures, we sorted thesesubsets by FACS to high purity, labeled them with distinctfluorescent dyes, remixed them, and followed their frequency

during culture. We did not observe any significant changes in thecomposition of the resulting cultures if costimulated with CD137or control antibody (Supplementary Fig. S1C). These resultssuggest that changes in their relative abundance do not explainchanges in DNA methylation.

CD137-elicited DNA-methylation changes correlate withimmune-gene expression

We decided to evaluate whether DNA-methylation changesobserved in our study altered mRNA transcription. To study thiscorrelation, a series of quantitative RT-PCR analyses were per-formed on the selected immune-relevant genes. For consistency inour analysis, experiments with an unrelated series of donors wereperformed under costimulation with urelumab (Fig. 3A) or with6B4 (Fig. 3B). CD8þ T-cell samples from each individual werepaired at each time point and a sufficient number of cases werestudied to mitigate the intrinsic genetic and epigenetic variabilityof human populations. Experiments studying protein expressionwere done when feasible (Fig. 4).

CD96mRNAwas consistently downregulated at all timepoints,including restimulation (Fig. 3). This surface molecule is consid-ered an important negative regulator of T and NK activationfollowing engagement to its ligand CD155 (32), whose blockade

Figure 1.

Screening for genes differentiallymethylated in CD8þ T cells upon CD137costimulation. A, Experimental tissueculture settingswith isolated peripheralblood CD8þ T cells seeded onto platescoated with anti-CD3e mAb (aCD3)with or without the anti-CD137 mAb(aCD 137, either urelumab or 6B4). Asindicated, T cells were transferred toantibody-free plates (resting lapse) inIL7-enriched media and transferred onday þ10 to anti–CD3e-coated plates.Samples were collected for differentpurposes at the indicated time pointsrepresented by arrows. B, Venndiagram showing the number of geneswith differential methylation patternsabove a cutoff of average delta-betavalues ¼ 0.25. Genes modified byurelumab and 6B4 are indicated. Thegenes significantly modified by bothagonist mAbs to CD137 are listed, withthose genes with known importantimmune functions highlighted in bold.

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enhances T and NK activation. Hence, we postulated that reducedexpression could result in enhanced activation and effector per-formance. We compared the intensity of surface CD96 expressionby FACS, andour resultswere in agreementwith this conclusion inthe majority of individual cases (Fig. 4A).

HHLA2 (B7-H5) is another emerging surface checkpoint recep-tor of the B7 family for T-cell activation, but in this case, mRNAexpression is clearly increased upon CD137 costimulation(Fig. 3A and B). The functional role of this surface molecule andits putative ligands remains obscure and controversial (33, 34).The absence of reliable detecting antibody reagents precludedanalysis of the protein in this case. However, the data suggest thatthis moiety is relevant for human T-cell biology even if notconserved in mice (33).

We found that themethylation status ofCCR5 andCXCR5wasmodified (Fig. 2), and such chemokine receptors were involvedin shaping CD8þ T-cell attraction to activated myeloid cells andfellow lymphocytes under inflammatory conditions. Reductionof CXCR5 mRNA and protein should mitigate the tendency ofsuch T cells to migrate to germinal centers, potentially leavingthem free to accomplish other tasks (35, 36), whereas enhancedexpression of CCR5 might make them more likely to meet andinteract with myeloid cells such as macrophages and dendriticcells. In this context, the function of CCR5 would also beenhanced by the concomitant upregulation observed withCCL5 (Figs. 3 and 4B), which is one of its ligands. Indeed,augmented CCL5 accumulation in the tissue culture superna-tant was readily observed upon restimulation of previously

CD137-costimulated CD8þ T cells (Fig. 4B). When these cul-ture supernatants were tested for their ability to attract mono-cyte-derived DCs, we found that CD137-costimulated super-natants were more powerful chemoattractants than theirrespective hIgG4 isotype control supernatants. This effect couldbe neutralized by a CCL5-blocking mAb (Fig. 4C).

The transcription factor TCF1 (encoded by TCF7) was dras-tically reduced (by as much as 85%) at the mRNA and proteinlevels by CD137 costimulation (Figs. 3 and 4A). This opens upan interesting area of research since the TCF1 transcriptionfactor, in conjunction with BCL6, seems to be critical in thecontrol of memory and stemness of T cells (37). Indeed, TCF1expression in T cells is associated with the exhausted phenotype(35, 37). The methylation status of the GFI-1 transcriptionalrepressor is also regulated by CD137 costimulation (Fig. 2Aand B), and this factor could mediate broader transcriptionaleffects on its target genes. Although its role in CD8þ T cellsremains poorly understood, key effects in Th17 and Th2 biol-ogy have been reported (38).

Controlling microRNA by gene methylation also constitutes amechanism to spread gene regulation to their targeted sequences.In our hands, MIR21 expression was decreased by DNA demeth-ylation at its locus (Fig. 5B and C).

DiscussionThis study shows that T-cell costimulation via CD137 (4-

1BB) results in epigenetic changes in the chromatin that affect

Figure 2.

Validation ofDNA-methylation changes in immune-relevant genes in a series of healthydonors. CD8þTcell cultures set as indicated in the diagramwere costimulatedwith urelumab (A) or 6B4 (B). The DNA-methylation changes compared with control were represented as a percentage of methylated sequences (analyzed bypyrosequencing) at the different timepoints; paired samples fromeach individual are linked by lines. Costimulationwith urelumab and6B4was performed in 9 and 10independent individual donors, respectively. Control and CD137-costimulated samples for each individual are linked by lines.






key genes playing a role in the ensuing immune response. Thesefindings may find application in understanding mechanisms ofaction of different immunotherapies and might be useful in thedevelopment of pharmacodynamics biomarkers. Our approachis based on simplistic culture systems in which primary humanCD8þ T lymphocytes are costimulated in the presence ofagonist antibodies and restimulated again to mimic antigenreexposure following a resting phase. Genome-wide DNA-methylation analysis using microarrays was chosen to explorethis subject. We focused on those genes whose attributedfunctions are predicted to affect T-cell immunity and thereforeCD137-based immunotherapy. Genes whose DNAmethylationis influenced by CD137 are broader than those on which wehave focused; however, our repeated findings conclusivelyindicated consistent regulation dependence on the CD137costimulation pathway.

Several studies integrating DNA-methylation profiles andgene expression data have shown that methylation at differentgenomic regions (promoters, gene bodies, or intergenicregions) is related to gene expression levels (28, 39). Althougha correlation between methylation and gene expression hasbeen observed in multiple studies, this scenario can be differentdepending on the methylation levels and the genomic regions.In this regard, correlation between gene body methylation and

expression was observed to be not only positive (40) ornegative (41) but also dependent on cell type (42). Hence, wehave explored changes in mRNA and, when feasible, proteinexpression.

CD96 is one of the consistently regulated genes that caught ourattention for its immune relevance as a checkpoint on T and NKcell activation. Even if the functional role of the observed changesremains to be determined, it is likely to be involved in rendering Tcells prone to activation. This is consistent with the observationsthat experimental lung metastases that result from the intrave-nous injection of tumor cells are decreased upon the attenuationof the CD96 coinhibitory pathway (43). As a result, CD96 is nowconsidered a very attractive immune checkpoint for immunother-apy (32).

Key to the performance of T cells is their ability to migrateup chemokine gradients or to produce chemokines that attractother leukocytes, encounters with whom could be functionallyimportant. Methylation changes to the CCR5, CXCR5, andCCL5 genes are very provocative and likely to affect cytotoxicT-lymphocyte (CTL) migration and homing, as well as favoringcertain cell-to-cell interactions amidst the tumor microenviron-ment or lymphoid tissue. Indeed, we observed that CCL5produced by CD137-costimulated CD8þ T cells attractedmonocyte-derived DCs.

Figure 3.

mRNA expression of the immune-relevant genes differentially methylated upon CD137 costimulation. Gene expression analyses by real-time PCR of the indicatedgenes were performed upon urelumab costimulation (A) in 12 individual samples or upon 6B4 (B) costimulation in 11 individual samples. Control and CD137-costimulated samples for each individual are linked by lines.

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In addition to chemotaxis, other important functions mayhave been overlooked in our analyses due to our imperfectknowledge of gene functions on the immune response. Forinstance, regulation of the inflammasome gene AIM2 (Fig. 2Aand B), which was reported in the AAI 2015meeting (44) as a keyregulator for CD4þ T-cell memory that is epigenetically regulatedby DNA methylation.

For gene-methylation regulation to be more effective, thetranscriptional control of other mechanisms that influence geneexpressionwould extend the effect to a broader list of downstreamgenes. Hence, control over transcription factors or microRNAs byepigenetic mechanisms is considered to be highly influential forgene-expression reprogramming. For this reason, we focused ontranscription factors and noncoding RNAs that could influencelong-term T-cell behavior. We found TCF1 and miR-21 to standout. Their influence on secondary target genes remains to be seen,but our findings open a novel layer of gene-expression regulationand a complex field to be explored.

TCF7 during chronic viral infections is known to be epige-netically regulated, resulting in changes in chromatin accessi-bility (45). TCF1 and CXCR5 are coordinately regulated inCD8þ T cells, which results in their differentiation into a subsetsimilar to follicular T helper cells (TFH like) in mice (35). Ourinterpretation is that CD137 costimulation leading to DNAmethylation changes would downregulate this TFH-like differ-entiation pattern. We have previously reported that human TFHcells are one of the only human lymphocyte subsets expressingbaseline CD137 in healthy conditions (14). Modulation ofCD137-dependent epigenetic changes in the TCF7 locus, whichencodes the TCF1 transcription factor, is of considerable inter-est, because this pathway orchestrates the stemness of T cells

(46), as well as their exhausted phenotype (35, 37, 46). Alongthis line of reasoning, TCF1 reduction could mitigate undesiredfunctional phenotypes of CD8þ T cells, thereby potentiallyenhancing tumor immunity.

Decreases in miR-21 could result in regulation of a number ofsecondary genes. MiR-21 is considered an onco-miR, because itpromotes tumor progression and turns on immunosuppressivemechanisms in colorectal cancer (46). However, little is knownabout how miR-21 would affect primary CD8þ T-cell differenti-ation and biology, although it has been reported to affect T-cellactivation (47, 48), apoptosis (49), and differentiation. OthermicroRNAs have been recognized as key factors in the regulationof CTL physiology (50).

The observed epigenetic changes do not only take place duringactivation, but persist, pointing to their role in shaping ultimateresponses upon antigen restimulation. Modifying DNA methyl-ation involves DNA replication and, therefore, we are likelyunderestimating epigenetic remodeling, because only part ofthe T-cell cultures may have divided a sufficient number of timesunder the influence of CD3 stimulation and CD137 costimula-tion. Because of methodological constraints, we have not sub-divided and isolated na€�ve and central memory peripheralblood CD8þ T cells, and the effects could vary depending on therelative abundance of each lymphocyte subset in the peripheralblood corresponding to each individual donor. To address thispoint, we set up costimulation cocultures with FACS-sorted pre-labeled na€�ve, memory, and effector cells. No significant differ-ences in proportions between CD137-costimulated and controlCD8þ T lymphocytes at the end of the 10-day cultures were found.However, changes in DNAmethylation induced by CD137 costi-mulation coincided with those that we reported comparing

Figure 4.

Changes in protein expression of the differentially methylated immune-relevant genes. A, Series of samples as in Fig. 3 and derived from 8 individuals that wereassessed for surface protein expression by immunofluorescence and flow cytometry. B, For CCL5 determination, cell culture supernatants of 9 independentrestimulated samples were removed and tested by ELISA. In C, supernatants of restimulated samples were tested for chemoattractant activity toward third-partymonocyte-derived DC, comparing urelumab and control antibody costimulated culture supernatants from (B; left). Right, the effects of a neutralizinganti-CCL5 mAb on recombinant CCL5 and the urelumab-costimulated supernatants are shown. Experiments were performed in triplicate with five independentlyraised DC cultures. Results are represented in a paired fashion and statistically compared with Wilcoxon tests for paired samples.






antigen-experienced with antigen-na€�ve CD8þ T cells. These find-ings reinforce the idea that CD137 costimulation sets a coursetoward memory differentiation, whose mechanistic underpin-nings are the subject of ongoing research.

Chromatin remodeling by epigenetic mechanisms of CD8þ Tcells modifies the expression of key genes upon T-cell activationanddifferentiation, for instance, during viral infection (51).CD28costimulation can also change the methylation of the IL2 pro-moter (52). Here, we connect the activity of a TNFR-familycostimulatory member to DNA methylation, which is a form oflong-term control of gene expression. The particular molecularpathways under CD137 control that lead to sequence-specificmethylation/demethylation of the target genes remain to beuncovered. Another implication of these data is that drugs mod-ifying DNAmethylation, such as DNMT inhibitors, would poten-tially modify CD137-based immunotherapy. In fact, modifica-tion of DNA-methylation affects genes relevant for Th1 biology invivo (53).

Application of our results and conclusions to CD137-tar-geted immunotherapy could still be premature. Our experi-ments were performed on resting peripheral blood CD8þ Tcells, whereas the main target of anti-CD137 in tumor-bearinghosts is proposed to be dysfunctional (exhausted) intratumoralT lymphocytes.

All in all, our study shows a new functional outcome followingCD137 costimulation. This costimulatory function is beingexploited for cancer immunotherapy with agonist antibodies(14) or chimeric antigen receptors encompassing the CD137cytoplasmic tail (54). Our results on epigenetic reprogrammingof cytotoxic T lymphocytes by CD137 costimulation imply far-reaching consequences to their functionality. Epigenetic repro-gramming is unique in the sense that would leave the T cells

poised to respond differentially when reencountering antigen inthe future.

Disclosure of Potential Conflicts of InterestI. Melero reports receiving a commercial research grant from Bristol-Myers

Squibb, Roche, and Alligator and is a consultant/advisory board member forBristol-Myers Squibb, Roche, Bayer, Tusk, AstraZeneca, Lilly, Alligator, andBioncotech.Nopotential conflicts of interestwere disclosedby theother authors.

Authors' ContributionsConception and design: M.A. Aznar, S. Labiano, A. Diaz-Lagares, J. Sandoval,I. MeleroDevelopment of methodology: M.A. Aznar, S. Labiano, A. Diaz-Lagares,C. Molina, M. Esteller, I. MeleroAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.A. Aznar, S. Labiano, A. Diaz-Lagares, C. Molina,A. Azpilikueta, I. Etxeberria, A.R. Sanchez-Paulete, M. Esteller, J. SandovalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.A. Aznar, S. Labiano, A. Diaz-Lagares, C. Molina,I. Etxeberria, M. Esteller, J. Sandoval, I. MeleroWriting, review, and/or revision of the manuscript: M.A. Aznar, S. Labiano,A. Diaz-Lagares, I. Etxeberria, M. Esteller, J. Sandoval, I. MeleroAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Garasa, A. Azpilikueta, A.R. Sanchez-Paulete,A.J. KormanStudy supervision: J. Sandoval, I. Melero

AcknowledgmentsI. Melero is supported by grants from MINECO (SAF2011-22831 and

SAF2014-52361-R), Departamento de Salud del Gobierno de Navarra, Redestem�aticas de investigaci�on cooperativa RETICC, European Commission VIIFramaework and Horizon 2020 programs (IACT and PROCROP), Fundaci�onde la Asociaci�on Espa~nola Contra el C�ancer (AECC), Fundaci�on BBVA andFundaci�on Caja Navarra. S. Labiano is the recipient of predoctoral scholarshipfrom MINECO. A. Diaz-Lagares is funded by Río Hortega Grant CM14/00067

Figure 5.

CD137 costimulation modifies methylation and expression of miR-21. A, Shows in green CpG islands in the MIR21 locus that were differentially methylated in thegenome-wide arraywith the corresponding delta values for urelumab costimulated samples.B,Validation by pyrosequencing ofmethylation changes in CD8þ T cellsfrom 8 individuals whose CD8þ T cells were costimulated by urelumab or control antibody and 8 individuals similarly costimulated by 6B4. C, RT-PCR expression ofmiR-21 in 11 individual donor CD8þ samples costimulated with urelumab or control antibody as indicated.

Aznar et al.





from ISCIII. J. Sandoval is funded by "Miguel Servet Program" from the FEDER,FSE, and ISCIII (CP13/00055) and a contribution from "Corte de Honor andFMV de 2011."

We are grateful to Drs. Alvaro Teijeira, Ana Rozaut, Jose Luis Perez-Gracia,Miguel Fernandez de Sanmamed, and Juan Jos�e Lasarte for helpful scientificdiscussions. We also appreciate technical support by Diana Garcia, CarlesArribas, and Elixabet Bola~nos. Navarrabiomed tissue bank nurses and medicalstaff are also gratefully acknowledged.

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 March 29, 2017; revised August 31, 2017; accepted November 3,2017; published OnlineFirst November 13, 2017.

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Aznar et al.




2018;6:69-78. Published OnlineFirst November 13, 2017.Cancer Immunol Res M. Angela Aznar, Sara Labiano, Angel Diaz-Lagares, et al.

Relevant Genes−Cell T +CD137 (4-1BB) Costimulation Modifies DNA Methylation in CD8

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