cd137 (4-1bb) costimulation modifies dna methylation in ... · 11/11/2017 · 31008 pamplona,...

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RESEARCH ARTICLE

CD137 (4-1BB) costimulation modifies DNA methylation in CD8+ T-cell relevant

genes.

M. Angela Aznar1*

, Sara Labiano1*

, Angel Diaz-Lagares2,3,7*

, Carmen Molina1, Saray

Garasa1, Arantza Azpilikueta

1, Iñaki Etxeberría

1, Alfonso R. Sanchez-Paulete

1, Alan J. Korman

4,

Manel Esteller2,5,6,7

, Juan Sandoval8**

and Ignacio Melero1,7**

.

1 Center for Applied Medical Research (CIMA), University of Navarra, E-31008 Pamplona, Spain. 2Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL),

L'Hospitalet de Llobregat, 08908 Barcelona, Catalonia, Spain. 3Current address: Translational Medical Oncology (Oncomet), Roche-CHUS Joint Unit, Health Research

Institute of Santiago (IDIS), University Clinical Hospital of Santiago (CHUS), 15706 Santiago de Compostela,

Galicia, Spain. 4Bristol-Myers Squibb, Redwood City, CA, USA.

5Departament de Ciències Fisiològiques II, Escola de Medicina, Universitat de Barcelona, 08907 Barcelona,

Catalonia, Spain; 6Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Catalonia, Spain

7 CIBERONC. Centro virtual de Investigacion Biomedica en red de Oncologia.

8 Biomarkers and Precision Medicine Unit (UByMP). Epigenomics core facility. Instituto Investigación

Sanitaria La Fe (IISlaFe), 46026 Valencia, Spain

Running title: CD137 costimulation and epigenetic reprogramming.

Keywords: CD137, 4-1BB, Costimulation, urelumab, DNA-methylation

Foot notes:*

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

** J. Sandoval, I. Melero will equally share credit for senior authorship.

Correspondence: Ignacio Melero MD PhD. CIMA. University of Navarra. Avenida Pio XII, 55.

31008 Pamplona, Spain. Phone +34948194700. [email protected] Financial support: IM. is supported by grants from MINECO (SAF2011-22831 and SAF2014-

52361-R), Departamento de Salud del Gobierno de Navarra, Redes temáticas de investigación

cooperativa RETICC, European Commission VII Framaework and Horizon 2020 programs (IACT

and PROCROP), Fundación de la Asociación Española Contra el Cáncer (AECC), Fundación

BBVA and Fundación Caja Navarra. S. L. is recipient of predoctoral scholarship from MINECO.

A.D-L. is funded by Río Hortega Grant CM14/00067 from ISCIII. J. S. is funded by “Miguel

Servet Program” from the FEDER, FSE and ISCIII (CP13/00055) and contribution from “Corte de

Honor and FMV de 2011” (to J.S.).

Conflict-of-interest disclosure: A.J.K. is a full time employee in Bristol Myers Squibb. I.M. has

served as a consultant for Bristol Myers Squibb, Roche Genentech, Alligator, Tusk, Boehringer,

Medimmune, Merck Serono

Word counts: Text: 3812 words; Figure/table count: 5 figures.

Research. on November 11, 2020. © 2017 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/2326-6066.CIR-17-0159

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/

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Abstract

CD137 (4-1BB) costimulation imprints long-term changes that instruct the ultimate behavior

of T cells that have previously experienced CD137 ligation. Epigenetic changes could provide a

suitable mechanism for these long-term consequences. Genome-wide DNA-methylation arrays

were carried out on human peripheral blood CD8+ T lymphocytes stimulated with agonist

monoclonal antibody to CD137, including urelumab, which is in phase I/II clinical trials for

cancer immunotherapy. Several genes showed consistent methylation patterns in response to

CD137 costimulation, which were confirmed 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. These genes

are also differentially methylated in naïve vs. antigen-experienced CD8+ T cells. The transcription

factor TCF1 and the microRNA miR-21 were regulated by DNA-methylation upon CD137

costimulation. Such gene-expression regulatory factors can, in turn, broaden the effects of DNA-

methylation by controlling expression of their target genes. Overall, chromatin remodeling is

postulated to leave CD137-costimulated T lymphocytes poised to differentially respond upon

subsequent antigen recognition. Accordingly, CD137 connects costimulation during priming to

genome-wide DNA methylation and chromatin reprogramming.




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Introduction

Costimulation 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 leave T cells poised to more robustly respond if

challenged at a later time point with antigen (2). Several gene expression control mechanisms may

be involved in such long-term regulation. Transcription factors do not adequately explain the long-

term effects, which suggests a role for epigenetic modifications (2,3). Epigenetic control of gene

expression acts as a switch to either induce or repress the transcriptional activity of multiple genes

implicated in different physiological and pathological conditions. Specific epigenetic mechanisms

have been identified as being responsible for regulating the expression of certain immune-related

genes (3,4). One of these epigenetic mechanisms is DNA-methylation, which consists of the

addition of a methyl group to the 5’ carbon of cytosine within cytosine-guanine dinucleotides

(CpGs). CpG DNA-methylation is considered perhaps the most fundamental molecular

phenomenon determining chromatin accessibility to the transcriptional machinery and thus leads

to gene expression regulation. Gene-specific DNA methylation is largely dependent on the activity

of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) that catalyze the transfer of a

methyl group from S-adenosyl methionine to DNA (5). These enzymes are drawn onto selective

genome locations to methylate cytosine bases in a sequence-specific fashion by poorly understood

targeting mechanisms. Such methylation patterns are subsequently inherited by daughter cells

following mitoses (6). Alterations in methylation patterns influence the balance of transcripts in

cells and contribute to pathological conditions such as cancer and the deregulation of the immune

system (7).

Critical loci in T lymphocytes are regulated by gene methylation and chromatin

accessibility, including the FOXP3 locus in natural Tregs (8), the PD-1 locus (PDCD1) in

exhausted T cells (9), and the differentiation to the IFNγ-producing phenotype under the influence




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of IL12 (10). How the epigenetic machinery selectively controls these phenomena in a gene-

specific manner remains poorly understood. Genome-wide approaches to resolving this issue have

seldom been undertaken (3,9).

In T-cell activation, costimulation via the TNFR family members is key to survival,

acquisition of effector functions, and memory differentiation (11-13). CD137 (4-1BB, TNFRSF9)

is not an exception (14). It gains surface expression on T cells only following TCR-mediated

priming (15), although its levels are augmented by CD28 costimulation (16). Ample experimental

evidence shows that when CD137 meets its ligand or agonist monoclonal antibodies (mAbs) on

CD8+ T cells, effector functions (17), survival (18), and memory generation are costimulated

(13,19-21). In this sense, agonist mAbs potentiate curative immune responses in mice bearing

tumors through immune mechanisms mediated primarily by CD8+ cytotoxic T lymphocytes

(14,22). In this regard, the fully human agonist mAbs to CD137, urelumab (23) and utomilumab

(24), are undergoing clinical trials as single agents or in combination with other

immunostimulatory mAbs (14). In rodent models CD137 engagement leads to more robust T-cell

responses, even when such mice are rechallenged with cognate antigen months after treatment

(25,26), indicating the need for long-term regulation mechanisms imprinted during the primary

response (14).

Hypothesizing that such long-term effects of CD137-ligation could be the consequence of

epigenetic changes encompassing chromatin remodeling, we performed experiments with

genome-wide high-throughput DNA-methylation arrays to identify immune genes on which

CD137 would influence cytosine methylation patterns at specific motifs. Using human primary

CD8+ T cells from series of healthy volunteers, the DNA-methylation changes were confirmed

and found to result in up- or down-regulation of mRNA and protein expression of such genes.




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Materials and Methods

T-cell isolation and T-cell culture.

Human CD8+ T lymphocytes were isolated from the peripheral blood of healthy donors by

Ficoll gradients, following a negative selection with CD8+ T Cell Isolation kit by in Automacs Pro

(Miltenyi Biotec). Blood samples were obtained from Navarra Blood and Tissue Bank.

Navarrabiomed Biobank, Navarra Health Department. CD8+ T lymphocytes were activated in 12-

well plates previously coated with anti-CD3ε (1μg/mL, clone OKT3) and anti-CD137 (10 μg/mL,

6B4 or urelumab) or respective isotype-matched control Ab (10 μg/mL) at 1.75 x 106 cells/well in

RPMI 1640 medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 100 IU/mL penicillin

and 100 g/mL streptomycin (Gibco) for 5 days (activation period). On day 5, T lymphocytes

were transferred onto 12-well plates in culture media supplemented with hIL7 (25 ng/μL,

Immunotools) for another five days (resting period). Restimulation was attained by transferring of

T cells to plates coated with anti-CD3ε mAb (1μg/mL, clone OKT3).

DNA extraction and Genome-wide DNA-methylation arrays.

DNA from CD8+ T lymphocytes was isolated using DNeasy Blood & Tissue kit (Sigma) and

quantified by Quant-iT™ PicoGreen dsDNA Reagent (Invitrogen). The integrity was analyzed in

a 1.3% agarose gel. Bisulfite conversion of 600 ng of each DNA sample was performed according

to the manufacturer's recommendation for Illumina Infinium Assay. Effective bisulfite conversion

was checked for three controls that were converted simultaneously with the samples. Four µl of

bisulfite converted DNA were used to hybridize on Infinium Human Methylation 450 BeadChip,

following the Illumina Infinium HD Methylation protocol. Chip analysis was performed using

Illumina HiScan SQ fluorescent scanner. The intensities of the images were extracted using




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GenomeStudio (2010.3) Methylation module (1.8.5) software (San Diego, California). The

methylation score of each CpG is represented as the beta (β) value.

The 450K DNA-methylation array by Illumina is an established, highly reproducible method

for DNA-methylation detection and has been validated in two independent laboratories (27). The

450K DNA-Methylation array includes 485,764 cytosine positions of the human genome that

were filtered by sex chromosome CpGs (avoiding sex link alterations) and non-valid CpGs (p-

value<0.001). The intensities of the images were extracted and normalized using GenomeStudio

(2011.1) Methylation module (1.9.0) software.

For determining differentially methylated CpGs an analysis using an absolute difference in

beta values of 0.25 and a standard deviation <0.1 were used for selecting the most relevant

positions.

Pyrosequencing.

Pyrosequencing analyses to determine CpG methylation status were developed as previously

described (28). Briefly, a minimum of 500 ng of DNA were converted using the EZ DNA-

methylation Gold (ZYMO RESEARCH) bisulfite conversion kit following the manufacturer’s

recommendations. Specific sets of primers for PCR amplification and sequencing were designed

using specific software (PyroMark assay design version 2.0.01.15). Primer sequences were

designed, when possible, to hybridize with CpG-free sites to ensure methylation-independent

amplification (see Supplementary Table 3). PCR was performed under standard conditions with

biotinylated primers and the PyroMark Vacuum Prep Tool (Biotage, Sweden) was used to prepare

single-stranded PCR products according to manufacturer’s instructions. PCR products were

observed on 2% agarose gels before pyrosequencing. Reactions were performed in a PyroMark

Q24 System version 2.0.6 (Qiagen) using appropriate reagents and protocols, and the methylation

value was obtained from the average of the CpG dinucleotides included in the sequence analyzed.




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Controls to assess correct bisulfite conversion of the DNA were included in each run, as well as

sequencing controls to ensure the reliability of the measurements. Graphic representation of

methylation values shows bars identifying CpG sites that present percentage methylation values.

RNA extraction and qRT-PCR.

T-lymphocyte samples were collected at activation, resting phase and restimulation time

points. Total RNA extraction was carried out by using Trizol (Invitrogen) following reverse

transcriptions with M-MLV reverse transcriptase (Invitrogen). Quantitative RT-PCR (qRT-PCR)

was performed with iQ SYBR green supermix in a CFX real time PCR detection system (Biorad).

Primer pairs used to detect gene expression are shown in Supplementary Table 3. Mature miR-21

expression was assessed by real-time PCR analysis using Taqman® microRNA assays (Life

technologies). Reverse transcription was performed on RNA using the TaqMan MicroRNA

Reverse Transcription Kit (cat no 4366596). Quantitative RT-PCR was performed with 2x

Taqman Fast Universal PCR Master Mix (cat no 4366072) according to manufacturer's protocol,

with specific TaqMan® primers. Gene and MIR21 expression data were normalized with levels of

the housekeeping gene H3 and RNU6B respectively, and represented according to this formula

2ΔCt (Ct H3 or RNU6 - Ct gene)

, where Ct corresponds to cycle number.

Flow cytometry and ELISA.

T lymphocytes were prestained with the Zombi NIR Fixable viability kit (Biolegend) as a

live/dead marker and pretreated with Beriglobin before staining. Surface staining was performed

with the 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 an isotype-matched negative control. The

True-Nuclear™ Transcription Factor Buffer Set (Biolegend) was used for intracellular staining of

TCF1 employing anti–TCF1-AF647 (7F11A10) and mouse IgG1-AF647 (MOPC-21), both




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purchased from Biolegend. Cell acquisition was carried out with FACSCanto II and FlowJo

(Treestar) software was used for data analysis.

CCL5 protein expression was measured from the supernatants of CD8+ T cell cultures using

the RANTES (CCL5) human SimpleStep ELISA kit (Abcam, ref AB174446).

Staining and culture of FACS sorted naïve, memory and effector CD8+ T cell subsets.

Human CD8+ T lymphocytes were immunomagnetically purified from peripheral blood of

healthy donors as described in Material and Methods. Subsequently, naïve and antigen-

experienced T-cell subsets were FACS-sorted as previously described (29). Briefly, CD8+ T cells

were pre-treated with Beriglobin before staining 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 Trace Violet (Invitrogen) or CFSE (BD) or left unstained and following pre-

labeling populations were remixed. Different combinations of these stainings were performed to

have cultures with each population stained with both cell dyes, rendering similar results. The

resulting remixed CD8+ T cells were activated with plate coated anti-CD3 mAb and costimulatory

mAb (Fig 1) in 96-well plates in identical density (cells/cm2) for five days. An identical

experimental procedure described in Materials and Methods section and depicted in Fig. 1 were

carried out.

Dendritic cell differentiation and chemotaxis assays.

CD14 cells from PBLs of healthy donors were purified with CD14 MicroBeads (Miltenyi) and

cultured in AIM V Medium (Gibco) with 1000u/mL of rhu-IL4 (R&D) and huGM-CSF (Bayer).

After six days, cells were collected, washed and seeded in 8µm pore polycarbonate transwell

(Costar), at 105 cells per well.




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The percent migration of dendritic cells (DC) to CCR5 was assessed towards recombinant

human CCL5 (Preprotech) (300 ng/mL) and towards supernatants derived from the 36h-

restimulated CD8+ T cells that had been previously activated in presence of either urelumab or

hIgG (from Fig. 4B). Following a 12h culture in the transwell setting, migrated DC of the bottom

chambers were recovered and counted. % Migration computes are refered to input DC. To block

CCL5-driven cell migration, DC were incubated for 1h with 0.8 µg/mL of human CCL5 blocking

antibody (R&D) before the migration assay. All the experiments were performed in triplicate.

Statistical analysis.

Prism software (GraphPad Software, Inc. La Jolla, Ca. USA) was used to analyze statistical

differences of absolute methylation level and mRNA and protein expression of target genes by

applying the paired Student’s t test, Mann Whitney U test or the Wilcoxon paired test. Values of *

P < 0.05, ** P < 0.01 and *** P < 0.001 were considered significant.




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Results

Agonist CD137 mAb regulates the DNA methylation of relevant CD8+ T-cell genes

Our initial hypothesis was that CD137 signaling in primed T lymphocytes would result in

chromatin remodeling through sequence-specific genomic DNA methylation. To study

modifications in DNA methylation at the genomic level, we used culture plate-bound CD3ε mAb

to mimic TCR priming together with urelumab (23) or 6B4 (30) as CD137 agonists (Fig. 1A).

CD8+ T cells were immunomagnetically isolated from the blood of healthy donor volunteers and

seeded onto plates coated with anti-CD3ε and anti-CD137. At the indicated points of time,

samples were retrieved to isolate genomic DNA, mRNA, or to measure protein expression in the

cells and supernatants. In the process of stimulation in culture, samples were transferred on day +5

to antibody-free plates to resemble a return-back to a resting status in the presence of the

homeostatic cytokine IL7. On day +10 of culture, T cells were stimulated again with solid-phase

bound anti-CD3ε as a surrogate of a subsequent antigen encounter. Samples were collected at a

series of time points following this secondary stimulation. This experimental approach sought to

observe changes caused by costimulation that would become imprinted into the chromatin of

CD137-costimulated CD8+ T cells. In order to assess nuclear gene DNA methylation in a genome-

wide fashion, the 450K DNA-methylation array was used in a series of CD8+ T cells from three

independent individuals costimulated by urelumab (or control IgG4 mAb), or by 6B4 acting on

CD8+ T cells from an additional unrelated donor. Differential methylation analyses were

performed at day +10, representing T lymphocytes in which the CD137-costimulation epigenetic

modifications would be already established, potentially leaving the cells poised to long-term

respond in a distinct manner.

At day +10 of CD8+ T-cell culture, 1028 differentially methylated CpGs corresponding to

907 genes (Fig. 1B) were found to be modified by CD137-costimulation in their methylation

status at specified CpGs, as determined by an unbiased bioinformatic analyses at consistent loci




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either after urelumab or 6B4 activation (Supplementary tables S1 and S2, respectively). As a

general tendency, most of the changes in CpGs whose methylation status was modified in

response to anti-CD137 mAb consisted of demethylation (87% CpGs for urelumab; 69% CpGs for

6B4) rather than hypermethylation. From the list of 52 genes which were differentially methylated

by CD137 costimulation observed both with urelumab and 6B4 (Fig. 1B), several genes (in bold)

attracted our attention for their well-described involvement in immune cell performance. This

hand-picked list was supported by PubMed publication searches and Gene Ontology (GO)

analyses, that showed an enrichment of immune response functions (GO:0006955, FDR =

0.0129). This group includes genes related to T-cell costimulation/coinhibition, inflammation and

inflammatory chemotaxis, immune cell transcription factors and the microRNA miR-21.

To confirm the reproducibility of these findings, we analyzed the identified methylated or

demethylated immune-relevant sequences by pyrosequencing in a series of unrelated individuals

(n = 19). As we hypothesized, these genes were modified in a consistent fashion with the findings

reported by the DNA-methylation microarrays, following both urelumab- or 6B4-elicited

costimulation (Fig. 2A and B).

DNA-methylation patterns are known to be different in naïve vs. antigen-experienced CD8+

T lymphocytes from human peripheral blood. DNA-methylation status of TCF7 and CCL5 loci,

which were identified in our genome-wide screenings (Fig. 1) as differentially methylated

following CD137 ligation, are indeed known to be differentially methylated in naïve vs. memory

CD8+ T cells (4,31). In fact, when we FACS-sorted naïve vs. memory and effector CD8

+ T cells

from peripheral blood (Supplementary Fig. S1A), we saw that our most relevant identified genes

followed similar DNA methylation pattern in peripheral blood naïve vs. memory CD8+ cells

(CD96, HHLA2, MIR21, and CXCR5) (Supplementary Fig. S1B).

To address whether the frequencies of originally naïve vs. memory cells changed in the

resulting cultures, we sorted these subsets by FACS to high-purity, labeled them with distinct




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fluorescent dyes, remixed them, and followed their frequency during culture. We did not observe

any significant changes in the composition of the resulting cultures if costimulated with CD137 or

control antibody (Supplementary Fig. S1C). These results suggest that changes in their relative

abundance do not explain changes in DNA-methylation.

CD137-elicited DNA-methylation changes correlate with immune-gene expression

We decided to evaluate whether DNA-methylation changes observed in our study altered

mRNA transcription. To study this correlation, a series of quantitative RT-PCR analyses were

performed on the selected immune-relevant genes. For consistency in our analysis, experiments

with an unrelated series of donors were performed under costimulation with urelumab (Fig. 3A) or

with 6B4 (Fig. 3B). CD8+ T-cell samples from each individual were paired at each time point and

a sufficient number of cases were studied to mitigate the intrinsic genetic and epigenetic

variability of human populations. Experiments studying protein expression were done when

feasible (Fig. 4).

CD96 mRNA was consistently down-regulated at all time points, including restimulation

(Fig. 3). This surface molecule is considered an important negative regulator of T and NK

activation following engagement to its ligand CD155 (32), whose blockade enhances T and NK

activation. Hence we postulated that reduced expression could result in enhanced activation and

effector performance. We compared the intensity of surface CD96 expression by FACS and our

results were in agreement with this conclusion in the majority of individual cases (Fig. 4A).

HHLA2 (B7-H5) is another emerging surface checkpoint receptor of the B7 family for T-cell

activation but in this case, mRNA expression is clearly increased upon CD137 costimulation (Fig.

3A and B). The functional role of this surface molecule and its putative ligands remains obscure

and controversial (33,34). The absence of reliable detecting antibody reagents precluded analysis




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of the protein in this case. However, the data suggest that this moiety is relevant for human T-cell

biology even if not conserved in mice (33).

We found that the methylation status of CCR5 and CXCR5 was modified (Fig. 2), and such

chemokine receptors were involved in shaping CD8+ T-cell attraction to activated myeloid cells

and fellow lymphocytes under inflammatory conditions. Reduction of CXCR5 mRNA and protein

should mitigate the tendency of such T cells to migrate to germinal centers, potentially leaving

them free to accomplish other tasks (35,36), whereas enhanced expression of CCR5 might make

them more likely to meet and interact with myeloid cells such as macrophages and dendritic cells.

In this context, the function of CCR5 would also be enhanced by the concomitant upregulation

observed with CCL5 (Figs. 3 and 4B), which is one of its ligands. Indeed, augmented CCL5

accumulation in the tissue culture supernatant was readily observed upon restimulation of

previously CD137-costimulated CD8+ T cells (Fig. 4B). When these culture supernatants were

tested for their ability to attract monocyte-derived DCs, we found that CD137-costimulated

supernatants were more powerful chemoattractants than their respective hIgG4 isotype control

supernatants. This effect could be neutralized by a CCL5-blocking mAb (Fig. 4C).

The transcription factor TCF1 (encoded by TCF7) was drastically reduced (by as much as

85%) at the mRNA and protein levels by CD137 costimulation (Figs. 3 and 4A). This opens up an

interesting area of research since the TCF1 transcription factor, in conjunction with BCL6, seems

to be critical in the control of memory and stemness of T cells (37). Indeed, TCF1 expression in T

cells is associated with the exhausted phenotype (35, 37). The methylation status of the GFI-1

transcriptional repressor is also regulated by CD137 costimulation (Fig. 2A and B) and this factor

could mediate broader transcriptional effects on its target genes. Although its role in CD8+ T cells

remains poorly understood, key effects in Th17 and Th2 biology have been reported (38).




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Controlling microRNA by gene methylation also constitutes a mechanism to spread gene

regulation to their targeted sequences. In our hands, MIR21 expression was decreased by DNA

demethylation at its locus (Fig. 5B and C).




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Discussion

This study shows that T-cell costimulation via CD137 (4-1BB) results in epigenetic changes

in the chromatin that affect key genes playing a role in the ensuing immune response. These

findings may find application in understanding mechanisms of action of different

immunotherapies and might be useful in the development of pharmacodynamics biomarkers. Our

approach is based on simplistic culture systems in which primary human CD8+ T lymphocytes are

costimulated in the presence of agonist antibodies and restimulated again to mimic antigen re-

exposure following a resting phase. Genome-wide DNA-methylation analysis using microarrays

was chosen to explore this subject. We focused on those genes whose attributed functions are

predicted to impact T-cell immunity and therefore CD137-based immunotherapy. Genes whose

DNA methylation is influenced by CD137 are broader than those on which we have focused,

however our repeated findings conclusively indicated consistent regulation-dependence on the

CD137 costimulation pathway.

Several studies integrating DNA-methylation profiles and gene expression data have shown

that methylation at different genomic regions (promoters, gene bodies, or intergenic regions) is

related to gene expression levels (28,39). Although a correlation between methylation and gene

expression has been observed in multiple studies, this scenario can be different depending 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) or negative (41) but also

dependent on cell type (42). Hence we have explored changes in mRNA and, when feasible,

protein expression.

CD96 is one of the consistently regulated genes that caught our attention for its immune

relevance as a checkpoint on T and NK cell activation. Even if the functional role of the observed

changes remain to be determined, it is likely to be involved in rendering T-cells prone to

activation. This is consistent with the observations that experimental lung metastases that result




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from the intravenous injection of tumor cells are decreased upon the attenuation of the CD96

coinhibitory pathway (43). As a result, CD96 is now considered a very attractive immune

checkpoint for immunotherapy (32).

Key to the performance of T cells is their ability to migrate up chemokine gradients or to

produce chemokines that attract other leukocytes, encounters with whom could be functionally

important. Methylation changes to the CCR5, CXCR5, and CCL5 genes are very provocative and

likely to affect cytotoxic T-lymphocyte (CTL) migration and homing, as well as favoring certain

cell-to-cell interactions amidst the tumor microenvironment or lymphoid tissue. Indeed, we

observed that CCL5 produced by CD137-costimulated CD8+ T cells attracted monocyte-derived

DC.

In addition to chemotaxis, other important functions may have been overlooked in our

analyses due to our imperfect knowledge of gene functions on the immune response. For instance,

regulation of the inflammasome gene AIM2 (Fig. 2A and B), which was reported in the AAI 2015

meeting (44) as a key regulator for CD4+ T-cell memory that is epigenetically regulated by DNA

methylation.

For gene-methylation regulation to be more effective, the transcriptional control of other

mechanisms that influence gene expression would extend the effect to a broader list of

downstream genes. Hence, control over transcription factors or microRNAs by epigenetic

mechanisms is considered to be highly influential for gene-expression reprogramming. For this

reason we focused on transcription factors and noncoding RNAs that could influence long-term T-

cell behavior. We found TCF1 and miR-21 to stand out. Their influence on secondary target genes

remains to be seen, but our findings open a novel layer of gene-expression regulation and a

complex field to be explored.




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TCF7 during chronic viral infections is known to be epigenetically regulated, resulting in

changes in chromatin accessibility (45). TCF1 and CXCR5 are coordinately regulated in CD8+ T

cells, which results in their differentiation into a subset similar to follicular T helper cells (TFH-

like) in mice (35). Our interpretation is that CD137 costimulation leading to DNA methylation

changes would downregulate this TFH-like differentiation pattern. We have previously reported

that human TFH cells are one of the only human lymphocyte subsets expressing baseline CD137 in

healthy conditions (14). Modulation of CD137-dependent epigenetic changes in the TCF7 locus,

which encodes the TCF1 transcription factor, is of considerable interest, since this pathway

orchestrates the stemness of T-cells (46), as well as their exhausted phenotype (35,37,46). Along

this line of reasoning, TCF1 reduction could mitigate undesired functional phenotypes of CD8+ T

cells, thereby potentially enhancing tumor immunity.

Decreases in miR-21 could result in regulation of a number of secondary genes. MiR-21 is

considered an onco-miR, because it promotes tumor progression and turns on immunosuppressive

mechanisms in colorectal cancer (46). However, little is known about how miR-21 would affect

primary CD8+ T-cell differentiation and biology, although it has been reported to affect T-cell

activation (47,48), apoptosis (49), and differentiation. Other microRNAs have been recognized as

key factors in the regulation of CTL physiology (50).

The observed epigenetic changes do not only take place during activation, but persist, pointing

to their role in shaping ultimate responses upon antigen restimulation. Modifying DNA

methylation involves DNA replication and, therefore, we are likely underestimating epigenetic

remodeling, since only part of the T-cell cultures may have divided a sufficient number of times

under the influence of CD3 stimulation and CD137 costimulation. Because of methodological

constraints, we have not subdivided and isolated naïve and central memory peripheral blood CD8+

T cells, and the effects could vary depending on the relative abundance of each lymphocyte subset

in the peripheral blood corresponding to each individual donor. To address this point, we set up




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costimulation cocultures with FACS-sorted pre-labeled naïve, memory, and effector cells. No

significant differences in proportions between CD137-costimulated and control CD8+ T

lymphocytes at the end of the 10-day cultures were found. However, changes in DNA methylation

induced by CD137 costimulation coincided with those that we reported comparing antigen-

experienced with antigen-naïve CD8+ T cells. These findings reinforce the idea that CD137

costimulation sets a course towards memory differentiation, whose mechanistic underpinnings are

the subject of ongoing research.

Chromatin remodeling by epigenetic mechanisms of CD8+ T cells modifies the expression of

key genes upon T-cell activation and differentiation, for instance during viral infection (51). CD28

costimulation can also change the methylation of the IL2 promoter (52). Here we connect the

activity of a TNFR-family costimulatory member to DNA methylation, which is a form of long-

term control of gene expression. The particular molecular pathways under CD137 control that lead

to sequence-specific methylation/demethylation of the target genes remain to be uncovered.

Another implication of these data is that drugs modifying DNA methylation, such as DNMT

inhibitors, would potentially modify CD137-based immunotherapy. In fact, modification of DNA-

methylation affects genes relevant for Th1 biology in vivo (53).

Application of our results and conclusions to CD137-targeted immunotherapy could still be

premature. Our experiments were performed on resting peripheral blood CD8+ T cells, whereas

the main target of anti-CD137 in tumor-bearing hosts is proposed to be dysfunctional (exhausted)

intratumoral T lymphocytes.

All in all, our study shows a new functional outcome following CD137 costimulation. This

costimulatory function is being exploited for cancer immunotherapy with agonist antibodies (14)

or chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic tail (54). Our results

on epigenetic reprogramming of cytotoxic T lymphocytes by CD137 costimulation imply far-




19

reaching consequences to their functionality. Epigenetic reprogramming is unique in the sense that

would leave the T cells poised to respond differentially when reencountering antigen in the future.




20

ACKNOWLEDGMENTS

We are grateful to Drs. Alvaro Teijeira, Ana Rozaut, Jose Luis Perez-Gracia, Miguel Fernandez de

Sanmamed and Juan José Lasarte for helpful scientific discussions. We also appreciate technical

support by Diana Garcia, Carles Arribas and Elixabet Bolaños. Navarrabiomed tissue bank nurses

and medical staff are also gratefully acknowledged.




21

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Figure legends

Figure 1. Screening for genes differentially methylated in CD8+ T cells upon CD137

costimulation. (A) Experimental tissue culture settings with isolated peripheral blood CD8+ T

cells seeded onto plates coated with anti-CD3ε mAb (αCD3) with or without the anti-CD137 mAb

(αCD 137, either urelumab or 6B4). As indicated, T cells were transferred to antibody-free plates

(resting lapse) in IL7-enriched media and transferred on day +10 to anti-CD3ε-coated plates.

Samples were collected for different purposes at the indicated timepoints represented by arrows.

(B) Venn diagram showing the number of genes with differential methylation patterns above a

cutoff of average delta-beta values = 0.25. Genes modified by urelumab and 6B4 are indicated.

The genes significantly modified by both agonist mAbs to CD137 are listed, with those genes with

known important immune functions highlighted in bold.

Figure 2. Validation of DNA-methylation changes in immune-relevant genes in a series

of healthy donors. CD8+ T cell cultures set as indicated in the diagram were costimulated with

urelumab (A) or 6B4 (B). The DNA-methylation changes compared to control were represented as

percentage of methylated sequences (analyzed by pyrosequencing) at the different time points,

paired samples from each individual are linked by lines. Costimulation with urelumab and 6B4

was performed in nine and ten independent individual donors respectively. Control and CD137-

costimulated samples for each individual are linked by lines.

Figure 3. mRNA expression of the immune-relevant genes differentially methylated

upon CD137 costimulation. Gene expression analyses by real-time PCR of the indicated genes

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.




25

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 were

assessed for surface protein expression by immunofluorescence and flow cytometry. (B) For

CCL5 determination, cell culture supernatants of nine independent restimulated samples were

removed and tested by ELISA. In (C), supernatants of restimulated samples were tested for

chemoattractant activity towards third party monocyte-derived DC, comparing urelumab and

control antibody costimulated culture supernatants from (B) (left graph). In the right panel, the

effects of a neutralizing anti-CCL5 mAb on recombinant CCL5 and the urelumab-costimulated

supernatants are shown. Experiments were performed in triplicate with five independently raised

DC cultures. Results are represented in a paired fashion and statistically compared with Wilcoxon

tests for paired samples.

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 the

genome-wide array with the corresponding delta values for urelumab costimulated samples. (B)

Validation by pyrosequencing of methylation changes in CD8+ T cells from eight individuals

whose CD8+ T cells were costimulated by urelumab or control antibody and eight individuals

similarly costimulated by 6B4. (C) RT-PCR expression of miR-21 in 11 individual donor CD8+

samples costimulated with urelumab or control antibody as indicated.




Figure 1




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Published OnlineFirst November 13, 2017.Cancer Immunol Res M. Angela Aznar, Sara Labiano, Angel Diaz-Lagares, et al. CD8+ T-cell relevant genes.CD137 (4-1BB) costimulation modifies DNA methylation in

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