hypomethylation of the treg-speciﬁc demethylated region in ... · atl. analysis of dna...

Research Article

Hypomethylation of the Treg-SpecificDemethylated Region in FOXP3 Is a Hallmarkof the Regulatory T-cell Subtype in AdultT-cell LeukemiaYayoi Shimazu1, Yutaka Shimazu1, Masakatsu Hishizawa1, Masahide Hamaguchi2,Yuya Nagai1, Noriko Sugino1, Sumie Fujii1, Masahiro Kawahara1, Norimitsu Kadowaki3,Hiroyoshi Nishikawa4, Shimon Sakaguchi5, and Akifumi Takaori-Kondo1

Abstract

Adult T-cell leukemia (ATL) is an aggressive T-cell malignancycaused by human T-cell leukemia virus type 1. Because ofits immunosuppressive property and resistance to treatment,patients with ATL have poor prognoses. ATL cells possess theregulatory T cell (Treg) phenotype, such as CD4 and CD25, andusually express forkhead box P3 (FOXP3). However, themechanisms of FOXP3 expression and its association withTreg-like characteristics in ATL remain unclear. Selective demeth-ylation of the Treg-specific demethylated region (TSDR) in theFOXP3 gene leads to stable FOXP3 expression and definesnatural Tregs. Here, we focus on the functional and clinicalrelationship between the epigenetic pattern of the TSDR and

ATL. Analysis of DNA methylation in specimens from 26patients with ATL showed that 15 patients (58%) hypomethy-lated the TSDR. The FOXP3þ cells were mainly observed in theTSDR-hypomethylated cases. The TSDR-hypomethylated ATLcells exerted more suppressive function than the TSDR-methyl-ated ATL cells. Thus, the epigenetic analysis of the FOXP3 geneidentified a distinct subtypewith Treg properties in heterogeneousATL. Furthermore,weobserved that thehypomethylationof TSDRwas associated with poor outcomes in ATL. These results suggestthat the DNA methylation status of the TSDR is an importanthallmark to define this heterogeneous disease and to predict ATLpatient prognosis. Cancer Immunol Res; 4(2); 136–45. �2015 AACR.

IntroductionAdult T-cell leukemia (ATL) is an aggressive T-cell malignancy

caused by human T-cell leukemia virus type 1 (HTLV-1; refs. 1, 2).HTLV-1 is endemic in southwest Japan, Sub-Saharan Africa, theCaribbean Basin, and South America (3). Approximately 5% ofHTLV-1–infected individuals developATL after a latency period of40 to 70 years from the initial HTLV-1 infection (3, 4). Theprognosis of ATL is usually poor with a median survival period

of approximately 1 year (5). The major obstacles in the treatmentof patients with ATL include resistance to a variety of cytotoxicagents and susceptibility to opportunistic infections because of itsimmunosuppressed property (3, 6, 7).

In HTLV-1–infected individuals, HTLV-1 proviral DNA hasbeen detected mainly in CD4þ T cells (8). However, humanCD4þ T cells are heterogeneous, and the subset of initialHTLV-1 infection in ATL patients remains to be clarified. MostATL cells express CD3, CD4, and CD25 (9). This phenotyperesembles that of regulatory T cells (Tregs), and several previousstudies have shown that 60% to 70% of ATL cases expressedforkhead box P3 (FOXP3; refs. 10, 11), which is amaster regulatorof Tregs (12).

Tregs play an important role in immune tolerance, and theyprevent the development of autoimmune and inflammatorydiseases by suppressing autoreactive T cells (13). FOXP3, a keytranscription factor, controls both the development of Tregs andtheir suppressive function (12–14). However, a substantialamount of evidence indicates that FOXP3 expression itself is notsufficient to establish the full phenotype and function of Tregs(15). In humans, FOXP3þ cells are heterogeneous and are notalways suppressive (16). Tregs are divided into two main sub-populations: natural or induced Tregs. The former is a genuineTreg, developing in the thymus at the stage of CD4 single-positivethymocytes (17, 18), whereas the latter is induced by differenti-ation in the periphery after antigenic stimulation (19, 20)and cannot fully exert suppressive activity (21, 22). SelectiveDNAhypomethylation of conserved cytosine-phosphate-guanine(CpG) residues within the FOXP3 locus, termed the Treg-specific

1Department of Hematology and Oncology, Graduate School of Med-icine, Kyoto University, Kyoto, Japan. 2Department of Endocrinologyand Metabolism, Graduate School of Medical Science, Kyoto Prefec-tural University of Medicine, Kyoto, Japan. 3Department of InternalMedicine, Division of Hematology, Rheumatology and RespiratoryMedicine, Faculty of Medicine, Kagawa University, Miki-cho, Kita-gun,Japan. 4Division of Cancer Immunology, Exploratory OncologyResearch and Clinical Trial Center (EPOC), National Cancer Center,Kashima, Japan. 5Department of Experimental Immunology, WorldPremier International Immunology Frontier Research Center, OsakaUniversity, Suita, Japan.

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

Y. Shimazu and Y. Shimazu contributed equally to this article.

Corresponding Author: Masakatsu Hishizawa, Graduate School of Medicine,Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507,Japan. Phone: 081-(0)75-751-4964; Fax: 081-(0)75-751-4963; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-15-0148

�2015 American Association for Cancer Research.

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demethylated region (TSDR), is important for distinguishingnatural Tregs from peripherally induced Tregs and for stabilizingFOXP3 expression in both humans and mice (23, 24).

Several studies have shown that FOXP3 expression variesamong ATL patients (10, 25), and that FOXP3-expressing ATLcells are not always suppressive (26). Therefore, we investigatedtheDNAmethylation pattern of the TSDR and its relevance to Tregproperties and clinical outcomes in ATL.

Materials and MethodsPrimary ATL cells and control cells

Primary ATL cells were obtained from 40 patients hospitalizedat Kyoto University Hospital or at affiliated hospitals during theperiod from 2001 to 2014. The diagnosis of ATL was based on theShimoyama classification (7). For CpG methylation analysis, weincludedonlymalepatients, because one allele of the TSDRon theFOXP3 locus of the X chromosome was methylated in females(24). The characteristics of 26 male patients are summarizedin Table 1 and Supplementary Table S1. Peripheral blood mono-nuclear cells (PBMC) were isolated with Ficoll-Paque (PharmaciaBiotech) density-gradient centrifugation. If the proportion ofabnormal cells in the PBMCs was less than 60%, we purified theATL cells by sorting (Supplementary Fig. S1). Control peripheralblood samples were obtained from 8 healthy volunteers. Thisstudy was performed in compliance with the Declaration ofHelsinki after approval by the Ethics Committee of Kyoto Uni-versity, Graduate School of Medicine. Written informed consentwas obtained from all patients and healthy volunteers.

Cell linesWe used HTLV-1–infected cell lines ATL-43T (27), MT-1 (28),

MJ (29), MT-2 (30), MT-4 (30), and Hut102 (31). ATL-43T werekindly provided by Dr. M. Maeda (Virus Institute, Kyoto Univer-sity, Japan) in 1991. MT-1, MT-2, and MT-4 were obtained fromJapanese Collection of Research Bioresources (JCRB) Cell Bank,and Hut102 and MJ were obtained from the American TypeCulture Collection between 2000 and 2014. All cell lines wereauthenticated using HTLV-1 clonal integration and tested formycoplasma contamination in 2014. These cell lines were cul-tured in RPMI-1640 medium (Nacalai Tesque) supplementedwith 10% FBS (Sigma-Aldrich) and 2 mmol/L penicillin, strep-

tomycin, and glutamine (Gibco BRL) at 37�C in a humidifiedincubator with 5% CO2 in air. For the ATL-43T cell line, which isIL2 dependent, the medium was supplemented with 0.5 nmol/Lrecombinant IL2 (Shionogi). All cell lines were not cultured forlonger than 1 month.

Bisulfite sequencingA CpG methylation analysis was performed as described with

slight modifications (23). Briefly, genomic DNA was isolatedfrom cells, and bisulfite treatment was performed using theMethylEasy Xceed Kit (Human Genetic Signatures). ModifiedDNA was amplified by PCR and cloned into a pGEM-T EasyVector System (Promega). The bisulfite sequencing–specific pri-mers are listed in Supplementary Table S2. The independentcolonies (16 colonies/region) were amplified with the IllustraTempliPhi Amplification Kit (GE Healthcare) and sequencedusing a 3130xl Genetic Analyzer (Applied Biosystems). The per-centage of methylation was calculated by dividing the number ofdemethylated colonies at the CpG site by 16.

qRT-PCR analysisTotal RNA was extracted from cells using an RNeasy Mini Kit

(Qiagen) and converted into cDNA using a SuperScript ReverseTranscriptase (Invitrogen) and Random Hexamers (Applied Bio-systems). qRT-PCRwas performed using the TaqMan gene expres-sion Kit (Applied Biosystems). The primers and probes for tax andHTLV-1 bZIP factor (HBZ) were as described (32, 33). Those forhuman FOXP3, CTLA4, HELIOS, EOS, b-ACTIN, and 18S rRNAwere purchased from Applied Biosystems. We calculated therelative mRNA expression based on the levels of expression inthe MJ cell line. All standards and samples were analyzed induplicate, and the average value was used for the calculations.

Flow cytometric analysisA phenotypic analysis was performed on the purified PBMCs.

The data were acquired using a FACS LSR II instrument (BDBioscience) or FACS Aria instrument (BD Bioscience) and ana-lyzed with FlowJo software (ver. 9.7.5; Tree Star, Inc.). For thesorting of primary ATL cells, cells were stained with anti-CD3(UCHT1; eBioscience), anti-CD4 (RPA-T4; eBioscience), anti-CD5 (UCH-T2; Biolegend), anti-CD7 (CD7-6B7; Biolegend),

Table 1. Patients' characteristics

Hypomethylatedpatients

Methylatedpatients P

Age (year), median (range) 57 (33–87) 54 (29–80)Clinical typeAcute 10 (66.7%) 7 (63.6%)Lymphoma 1 (6.7%) 2 (18.1%)Chronic 3 (20.0%) 1 (9.0%)Smoldering 1 (6.7%) 1 (9.0%)

Alb (g/dL), median (range) 3.9 (2.3–4.4) 3.6 (2.6–4.5) 0.63LDH (IU/L), median (range) 1,037 (323–6,451) 485 (195–3,092) 0.46Ca (mg/dL), median (range) 9.7 (8.8–16.9) 11.2 (9.1–16.9) 0.38IgG (mg/dL), median (range) 1,037 (323–6,451) 990 (624–1,460) 0.80sIL2R (U/mL), median (range) 30,071 (758–104,000) 18,670 (1,220–232,000) 0.55OS (days), median (range) 283 (16–3,099) 627 (116–3,923) 0.1Cause of deathATL 8 (66.6%) 5 (71.4%)Infection 2 (16.7%) 2 (28.6%)Others 2 (16.7%) 0

Abbreviation: sIL2R, soluble IL2 receptor.

Epigenetic Alterations of the FOXP3 Gene in ATL

www.aacrjournals.org Cancer Immunol Res; 4(2) February 2016 137




anti-CD25 (M-A251; BD Bioscience), and LIVE/DEAD FixableDeadCell StainKits (Invitrogen). Cellswere sortedwith FACSAria(BD Bioscience) and used for further experiments (Supplemen-tary Fig. S1).

Functional assay to evaluate the suppressive activity of ATL cellsin vitro

The suppressive function of ATL cells was assayed using amodified protocol (16). Briefly, CD4þCD25�CD45RAþ T cellsfrom a healthy donor were used as responder cells and stainedwith theCell Trace Violet Cell Proliferation Kit (Molecular Probes,Life Technologies) according to the manufacturer's instructions.CD4�CD8� cellswere used as antigen-presenting cells (APC) afterirradiation (18.5 Gy). Responder cells were cocultured with thesamenumbers of ATL cells with 10-fold APCs and stimulatedwith0.5 mg/mL soluble anti-CD3 (eBioscience) and 0.1 mg/mL solubleanti-CD28 (eBioscience) in supplemented RPMI medium. Thenumber of proliferated violet-labeled cells was assessed by flowcytometry after culturing for 84 to 90 hours.

Cytokine stainingCytokine staining was performed as described (16). Briefly,

purified T cells were stimulated by phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and ionomycin (Sigma-Aldrich)for 4 hours in the presence of a Golgi transport inhibitor, mon-ensin (GolgiStop; BDPharmingen). After stainingwith antibodiesto surface moieties, the cells were fixed and permeabilized byCytofix/Cytoperm and PermWash (BDPharmingen) according tothemanufacturer's instructions. Cells were then stainedwith anti-cytokine or isotype-matched antibodies and analyzed by flowcytometry.

Statistical analysisStatistical analyses were performed using Graph Pad Prism

6.0 (Graph Pad Software). Survival curves of the ATL patientswere calculated by the Kaplan–Meier method using the SPSSStatistics software program, version 22.0 (IBM). Differencesbetween two groups were evaluated by unpaired t tests. Forthe comparison of multiple groups, we used the Tukey method.Comparisons of overall survival (OS) were carried out betweentwo groups with two-sided log-rank tests. P values < 0.05 wereconsidered significant.

ResultsMethylation status of FOXP3, CTLA4, and HELIOS genesin ATL cells

We first investigated the DNA methylation status of CpGresidues in the FOXP3 gene locus from amplicons (amp) 1 to11 by using the bisulfite sequencing method, based on aprevious report (24). The MJ cell line and ATL cells from case2 showed hypomethylation states of FOXP3 that were similar tothose of Tregs, whereas the MT-1 cell line and ATL cells fromcase 17 showed methylation states similar to those of CD4þ

CD25� conventional T cells (Fig. 1A). Subsequently, we ana-lyzed the DNA methylation status of amp 5 of FOXP3 by thesame method, because this region, referred to as the TSDR, isimportant in the development and function of Tregs (24). Wecalculated the mean methylation percentage in the TSDR. A ratelower than 50% was considered hypomethylated, and greaterthan 50% was considered methylated. ATL cells from 15 (58%)of the 26 patients analyzed have hypomethylation states sim-

ilar to normal Tregs (Fig. 1B, left). The median methylation rateof the TSDR-hypomethylated cases and that of the TSDR-methylated cases were 15% (range, 0%–40%) and 85% (range,73%–96%), respectively.

The CTLA4 and HELIOS regions are also differentially meth-ylated in Tregs and CD4þCD25� conventional T cells (23). Weconfirmed that the CTLA4 exon 2 and HELIOS intron 5 regionswere hypomethylated in normal Tregs, but highly methylated inCD4þCD25� conventional T cells (Supplementary Fig. S2).Therefore, we examined the DNA methylation of both of theseareas in primary ATL cells and found that they were both pre-dominantly hypomethylated, except for 4 cases in CTLA4 exon 2and 1 case in HELIOS intron 5. All cases with CTLA4 exon 2methylation orHELIOS intron5methylationwere included in theTSDR-methylated cases.

DNA methylation of the TSDR, FOXP3 expression, and Tregsignatures

We analyzed the expression of Treg-related genes and variouscytokines. With the use of flow cytometric analysis, FOXP3expression was observed in almost all of the patients identifiedwith a hypomethylated state. The percentage of FOXP3þ cells wassignificantly higher in the TSDR-hypomethylated cases than in themethylated cases (Fig. 2A and Supplementary Fig. S3A). Themeanfluorescence intensity (MFI) of FOXP3 tended to be higher in thehypomethylated cases, but the difference was statistically mar-ginal. Transcriptional analysis by qRT-PCR presented a trend ofmore FOXP3 mRNA in the TSDR-hypomethylated cases (Fig. 2Band Supplementary Fig. S3B). We could not exclude the possi-bility that contamination of the normal Treg population mightlead to the elevation of FOXP3 expression in whole PBMCsamples, so we isolated the ATL cells by sorting. Purified ATLcells from the TSDR-hypomethylated cases had significantlymoreFOXP3mRNA comparedwith themethylated cases (P¼ 0.01; Fig.2B and Supplementary Fig. S3C).

In order to further characterize the ATL cells, we subdividedTregs into three fractions (fractions I, II, and III) according to theexpression of CD45RA and FOXP3, as described (16). We ana-lyzed 8 TSDR-hypomethylated cases and 8 methylated cases. ATLcells from the TSDR-hypomethylated cases were mainly classifiedinto fraction II or fraction III, whereas those from the TSDR-methylated cases belonged to various subsets (Fig. 3). In most ofthe TSDR-hypomethylated cases, CD45RA�FOXP3high–activatedTregs constituted a proportion of the ATL cells. ATL cells from case8, which had about 40% methylation of the TSDR, also wereprimarily in fraction II.

The expression of other Treg-related proteins besides FOXP3,such as CD25, CTLA4, GITR, CCR4, andHELIOS, was analyzed in14 ATL patients, including 8 TSDR-hypomethylated cases (Sup-plementary Fig. S4). Most patients had expression of CD25,CTLA4, GITR, CCR4, and HELIOS, consistent with the phenotypeof Tregs. However, the TSDR-hypomethylated and -methylatedcases were not significantly different (Supplementary Fig. S4A–S4E). In addition, expression of proliferation marker Ki67 (16)was not significantly different between the groups (Supplemen-tary Fig. S4F).

We analyzed cytokine secretion from the ATL cells of 14patients whose cells were stimulated with PMA and ionomycin(Supplementary Fig. S5). The cells secreted low amounts ofIFNg , IL4, IL17, IL10, IL2, and TNFa with no differencesbetween the TSDR-hypomethylated and -methylated cases.

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Cancer Immunol Res; 4(2) February 2016 Cancer Immunology Research138




Expression of mRNA from CTLA4, HELIOS, and EOS, whichforms a complex with FOXP3 (34), in the cells of 25 ATLpatients varied among the ATL cases, with no significant differ-ences in methylation status of TSDR (Supplementary Fig. S6).Together, these results indicated that the common character-istics of Tregs (e.g., FOXP3 expression, cell surface phenotype,and cytokine secretion) were indicated by hypomethylation ofthe TSDR in ATL cells. However, only FOXP3 expression coulddiscriminate between the TSDR-hypomethylated and -methyl-ated cases.

Functional analysis of suppressive activity of primary ATL cellsin vitro

To clarify the functional differences between the TSDR-hypo-methylated and the -methylated cases, we measured suppres-sive activity, a major characteristic of Tregs. We analyzed 10 ATLcells (5 TSDR-hypomethylated cases and 5 TSDR-methylatedcases). When we cocultured healthy CD4þCD45RAþ na€�ve Tcells as responders with the TSDR-hypomethylated ATL cells aseffectors, the proliferative activity and number of responder Tcells were suppressed (Fig. 4). In contrast, the TSDR-methylated

Figure 1.The TSDR of ATL cells exhibits Treg-like hypomethylated status or CD4þ

conventional T cell–like methylatedstatus. Bisulfite sequencing wasperformed using genomic DNA fromeach sample. The frequency of thedemethylation of CpG is shown.Yellow, hypomethylated; and blue,methylated in each CpG. A, the DNAmethylation status in the TSDR of theHTLV-1–infected cell lines and primaryATL cells was analyzed, and all TSDR,from amplicons (amp) 1 to 11, areillustrated. MJ cell line was used as theTSDR-hypomethylatedrepresentative, and MT-1 cell line wasused as the TSDR-methylatedrepresentative. For primary ATL cells,case 2 was used as the TSDR-hypomethylated representative, andcase 17 was used as the TSDR-methylated representative. B, theDNAmethylation status of the TSDR of ahealthy donor and primary ATL cells.Tregs from a healthy donor were usedas the TSDR-hypomethylated control,and CD4þCD25� conventional T cellsfrom a healthy donor were used as theTSDR-methylated control. Thenumber of primary ATL cellscorresponds to each case numbershown in Supplementary Table S1. Thedata shown are the sum of threeindependent experiments.






ATL cells could not suppress. Similar results were seen in theHTLV-1–infected cell lines (Supplementary Fig. S7). Thus, themethylation status of the TSDR was associated with suppressiveactivity.

HTLV-1–encoded tax and HBZ not related to thehypomethylation of TSDR

It was reported that the HTLV-1–associated molecules taxand HBZ affected the expression of FOXP3 (35, 36). To clarifythe relationship between FOXP3 expression and these mole-cules, we analyzed mRNA expression. Consistent with previousreports (37), ATL cells contained little or no tax mRNA. Themethylation status of the TSDR was not related to the amount

of tax mRNA (Supplementary Fig. S8A). HBZ mRNA was alsoindependent of the methylation status of the TSDR in ATLpatients (Supplementary Fig. S8B). Taken together, theseresults show that there is no obvious relation betweenHTLV-1 viral molecules and the methylation pattern of theTSDR in ATL.

Hypomethylation of TSDR as a predictive factor for survivalin ATL

To assess the clinical significance of the epigenetic statusof FOXP3, we divided 25 patients into two groups accordingto the TSDR methylation status. One patient (case 5) wasexcluded in this analysis because of missing data. Clinical

AC

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Figure 2.The hypomethylation of the TSDRcorrelates with FOXP3 expression. A,primary ATL cells were stained withFOXP3 antibody, and the expressionlevel of FOXP3 was assessed by flowcytometry. Representative histogramdata of FOXP3 expression areshown. Case 13 and case 22 arerepresentatives of thehypomethylated TSDR(hypomethylation) and themethylated TSDR (methylation),respectively. The frequency ofFOXP3þ cells and MFI of FOXP3 werecalculated and plotted. Each dotrepresents individual ATL cells fromeach patient, and the bar indicates themedian. B, the FOXP3mRNA levels ofprimary ATL cells were assessed byqRT-PCR. The FOXP3mRNA level wasdivided by the b-actin mRNA level.The relative mRNA levels werecalculated based on the expression ofmRNA in the MJ cell line. Bars indicatethe median. Bold circles and squaresindicate sorted samples. Solid circlesand squares indicate unsortedsamples. Statistical analysis wasperformed, and P values are shownin the figure.

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characteristics including the median age, clinical type, historyof stem cell transplantation (SCT), and putative prognosticfactors, such as concentrations of serum albumin, lactatedehydrogenase (LDH), calcium, IgG, and soluble IL2 receptor,are not significantly different between the TSDR-hypomethy-lated group and the TSDR-methylated group (Table 1 andSupplementary Table S1). The median OS for the entire groupwas 315 days (range, 16–3,923 days), and 19 of the 25 patients(76%) died. The median OS of the TSDR-hypomethylatedgroup and that of the TSDR-methylated group were 283 days(range, 16–3,099 days) and 627 days (range, 116–3,923 days),

respectively (Supplementary Fig. S9A). As the patients withchronic and smoldering types of ATL had better prognosesthan those with the acute or lymphoma types, we excludedboth of these clinical types and reanalyzed OS. The TSDR-hypomethylated group had significantly inferior OS comparedwith the TSDR-methylated group: TSDR-hypomethylated,median OS of 186 days (range, 16–691 days); TSDR-methyl-ated, median of 627 days (range, 116–3,923 days), P ¼ 0.02(Fig. 5). Of the 10 deceased patients in the TSDR-hypomethy-lated group, 8 patients succumbed to ATL, 1 patient to infec-tion, and 1 to treatment-related mortality. Of the 6 deceased


Healthy donor

Fr I: 3.1%

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Figure 3.Subpopulation of FOXP3þ ATL cells.The expression of CD45RA andFOXP3 of primary ATL cells isshown. CD45RA, y-axis; FOXP3,x-axis. We analyzed a total of 16 cases(8 TSDR-hypomethylated casesand 8 methylated cases). Therepresentative data of a healthyindividual are also shown. Asmentioned in a previous study (16),Tregs can be divided into three groupsaccording to their function: fractionsI, II, and III. Each square indicatesfraction I (Fr I), fraction II (Fr II), andfraction III (Fr III), respectively.Numbers indicate the frequency ofCD4þ T cells in each fraction.






patients in the TSDR-methylated group, 4 patients succumbedto ATL and 2 to infection (Supplementary Table S1). Theseresults suggest that the methylation pattern of TSDR could beassociated with survival for ATL patients.

Next, we analyzed the relationship between the expressionof FOXP3 and survival. Although the high frequency ofCD45RA�FOXP3high T cells had a tendency to reduce survival,this association was not statistically significant (P ¼ 0.14;

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Figure 4.Primary ATL cells with thehypomethylated TSDR showedsuppressive function resembling Treg.CD4þCD45RAþ na€�ve T cells from ahealthy donor were used as respondercells, cultured with irradiated APC withor without primary ATL cells frompatients. Responder cells and primaryATL cells were added at a 1:1 ratio. ATLcells from cases 5 and 17 were used asrepresentative TSDR-hypomethylatedcases (hypomethylation) and TSDR-methylated cases (methylation),respectively. The proliferation ofresponder cells was assessed by thedilution of labeled dye. Representativedata of responder cells alone, ATL cellsfrom the hypomethylated TSDR added,and ATL cells from the methylatedTSDR are added (top). The numbersindicate the frequency of proliferatingresponder cells. The percentage ofproliferating responder cells (middle)and the numbers of proliferating cells(bottom) were plotted as mean � SD.Representative data are shown, as theexperiments were independentlyperformed three times using differentATL cells with similar results. P valuesare indicated in each part of thefigure; NS, not statistically significant.

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Supplementary Fig. S9B). Even when we include 14 femalepatients for the analysis, the difference was not apparent (P ¼0.16; Supplementary Figs. S9C and S10; Supplementary Table S3).Thus, the methylation pattern of the TSDR may provide moreprecise prognostic stratification than expression of FOXP3.

DiscussionIn this study, we found that ATL cells contain a unique cell

subtype with a Treg-like hypomethylation status in the TSDR. TheTSDR-hypomethylated ATL cells not only had higher expressionof FOXP3 and more suppressive activity, but also poorer clinicaloutcomes.

FOXP3 expression in ATL cell lines and primary ATL cells hasbeen investigated by many researchers at the mRNA and proteinlevels, and it varies widely among cell lines and individual cases(10, 11, 25, 26, 38, 39). Our data also showed a wide range ofFOXP3 expression among primary ATL cells. Among these het-erogeneous clusters, we could differentially identify one specificsubtype, which shows the TSDR-hypomethylated status is similarto that of natural Tregs. These TSDR-hypomethylated ATL cellsresemble natural Tregs not only in their expression of FOXP3, butalso other Treg-specific markers, such as CD25, CTLA4, HELIOS,and GITR. However, the TSDR-methylated ATL cells alsoexpressed Treg-specific markers other than FOXP3. In conven-tional T cells, the expression of CD25 and CTLA4 is readilyinduced by T-cell activation, irrespective of their DNA methyla-tion status (23). This may explain the expression of such mole-cules in ATL cells with a methylated TSDR.

The suppressive function of ATL cells has also been a subject ofconsiderable discussion. Some studies showed suppressive activ-ity of ATL cells (39, 40), but others did not (26).We demonstratedthat the TSDR-hypomethylated ATL cells, which showed higherFOXP3 expression, had suppressive function, whereas the TSDR-methylated ATL cells, which had less FOXP3 expression, did not.In natural Tregs, the DNA hypomethylation status of the TSDR isessential for stable FOXP3 expression and suppressive function(24, 41, 42). Similarly, in ATL cells, we found that the DNAhypomethylation status of the TSDR was closely associated withFOXP3 expression and function.

The HTLV-1–encoded genes, tax and HBZ, can modulate theexpression of FOXP3. Transfection of tax DNA to CD4þCD25þ

T cells resulted in a reduction of FOXP3 mRNA and suppressivefunction (35). In HBZ transgenic mice, the number of FOXP3þ

CD4þ T cells increased, and these HBZ-induced Tregs had unsta-ble FOXP3 expression, impaired function, andmethylated TSDRs(36). However, primary ATL cells were not assessed in theseexperiments. In the present study, no correlation was detectedbetween HTLV-1–associated molecules and the expression ofFOXP3 or the methylation status of the TSDR.

Several studies have tried to identify the origin of ATL cells(11, 43), but the answer as to whether the origin of ATL cells isnatural Tregs or conventional T cells has remained controversial. TheDNA demethylation of the FOXP3 gene is thought to be a specificphenomenon of natural Tregs. In addition, the HTLV-1 gene itselfcould change FOXP3 expression through the TGFb signal pathway(44), but such FOXP3þ T cells are induced Tregs, not natural Tregs.We therefore speculate that the demethylation of the TSDR in ATLcells is not a secondaryevent afterHTLV-1 infection, and that theATLcells with the hypomethylated TSDR may be derived from naturalTregs. However, we cannot exclude the possibilities that (i) theHTLV-1 virus may have infected CD4þFOXP3� non-Treg cells and(ii) epigenetic change had occurred during the development of ATL.Therefore, we cannot draw any conclusions about the origin of ATLcells from the findings of the present study. More detailed char-acterizationsof theTSDR-hypomethylatedATL cells at different timepoints during ATL progression would provide useful informationabout the etiology of ATL cells.

ATL is divided into four clinical variants: acute, lymphoma,chronic, and smoldering. The former two variants have a moreaggressive clinical course and shorter OS than the latter two. TheATL-prognostic index was recently proposed to identify the clin-ical risk among acute and lymphoma-type patients (45). Untilnow, the biologic phenotype could not be used to distinguish theprognosis (46).

With respect to FOXP3 expression, some studies have evaluatedATL cells by immunohistochemical staining (25, 47) or qRT-PCR(48), but these studies did not show a correlation with OS.Similarly, we could not show a significant association betweenFOXP3 expression and OS. However, the results of the presentstudy demonstrate that the methylation pattern of the TSDR iscorrelated withOS, regardless of the treatment with SCT (data notshown), and it was maintained when we omitted TSDR-methyl-ated but FOXP3þ cases (data not shown). The hypomethylationof the TSDR in ATL cells led to FOXP3 expression, but even theTSDR-methylated cases often expressed FOXP3 (Figs. 2 and 3).Thehypomethylationof the TSDR inATL cells is closely associatedwith their suppressive function, like that of natural Tregs. Incontrast, the expression of FOXP3 is not specific to Tregs, andthe FOXP3-expressing ATL cells with themethylated TSDRmaybeclassifiedwithnon-Tregs that donot possess suppressive function.Such functional deference according to the methylation statusmight influence the clinical course of ATL. However, the intrinsicimmunosuppression in the TSDR-hypomethylated patientsseems to be unrelated to the cause of death in the present smallcohort, and we could not elucidate the reason for their poorsurvival. The possibility of other causes, such as drug resistance,will be a subject for future investigation.

The limitation of our study was that we could only analyze 26cases of ATL regarding themethylation pattern of the TSDR, and thetreatment strategies differed among these patients. It is necessary toconfirm our results by conducting a large-scale prospective clinicalstudy. Inaddition,we couldnot evaluate themethylationpatternsof

Days after diagnosis

OS


1.0

0.8

0.6

0.4

0.2

0

P = 0.02

4,0003,0001,000 2,0000

Figure 5.OS according to the methylation status of the TSDR. Survival curves of thehypomethylated TSDR (solid line) and the methylated TSDR (dotted line)were calculated by the Kaplan–Meier method. P value is shown in the figure.






the TSDR inwomen.We analyzedother Treg-specific demethylationregions, e.g., CTLA4 exon 2 and HELIOS intron 5, but the hypo-methylationof such regionswasnot specific toATLpatientswith thehypomethylated TSDR. Therefore, to analyze female cases, otherassessment strategies are needed. In the male transplant setting, itsprognosis is worse than in females (49). We do not know whetherour results could be applied to cases involving women. To date, thedefinition of hypomethylation is not standardized. We tentativelydefined the cutoff values for FOXP3DNAhypomethylation as 50%.According to this definition, we can classify the hypomethylatedcases with high FOXP3 expression in this small cohort, but therelevance of this threshold should be reevaluated with amore large-scale study.However, we believe that it is important to recognize theunique ATL subtype defined according to the hypomethylationstatus of the TSDR. In the future, different treatment strategiesmightbe needed for this poor-prognosis ATL subtype.

In conclusion, we propose that a distinct subtype of theheterogeneous ATL disease can be defined according to thehypomethylated status of the TSDR. This Treg subtype is relatedto the biologic phenotype and clinical course of ATL. The hypo-methylated status of the TSDR in ATL cells might be a promisingtool for risk classification.

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

Authors' ContributionsConception and design: Y. Shimazu, Y. Shimazu,M. Hishizawa,M.Hamaguchi,N. Kadowaki, A. Takaori-KondoDevelopment of methodology: Y. Shimazu, Y. Shimazu, S. Sakaguchi

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Shimazu, Y. Shimazu, M. Hishizawa, Y. Nagai,N. Sugino, S. FujiiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):Y. Shimazu, Y. Shimazu,M.Hishizawa,M. Kawahara,H. Nishikawa, A. Takaori-KondoWriting, review, and/or revision of the manuscript: Y. Shimazu, Y. Shimazu,M. Hishizawa, M. Hamaguchi, M. Kawahara, A. Takaori-KondoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Shimazu, M. Kawahara, H. Nishikawa,A. Takaori-KondoStudy supervision:M.Hishizawa,N. Kadowaki, S. Sakaguchi, A. Takaori-Kondo

AcknowledgmentsThe authors thank Dr. Maeda (Virus Institute, Kyoto University, Japan) for

providing ATL-43T. They also thank clinicians in Japanese RedCrossWakayamaMedical Center (Wakayama, Japan), Kitano Hospital (Osaka, Japan), KokuraMemorial Hospital (Kokura, Japan), Kurashiki Central Hospital (Kurashiki,Japan), Kyoto City Hospital (Kyoto, Japan), Osaka Red Cross Hospital (Osaka,Japan), Otsu Red Cross Hospital (Otsu, Japan), Shinko Hospital (Kobe, Japan),Tenri Hospital (Tenri, Japan), and The Japan Baptist Hospital (Kyoto, Japan) forproviding clinical samples.

Grant SupportThis work was supported by grant-in-aid for Scientific Research

(C; 24591392; M. Hishizawa) from the Ministry of Education, Culture, Sports,Science and Technology of Japan.

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 June 16, 2015; revised November 10, 2015; accepted November 26,2015; published OnlineFirst December 17, 2015.

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2016;4:136-145. Published OnlineFirst December 17, 2015.Cancer Immunol Res Yayoi Shimazu, Yutaka Shimazu, Masakatsu Hishizawa, et al. T-cell Leukemia

Is a Hallmark of the Regulatory T-cell Subtype in AdultFOXP3Hypomethylation of the Treg-Specific Demethylated Region in

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