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Hypomethylation of the Treg-specific demethylated region in FOXP3 is a

hallmark of the regulatory T-cell subtype in adult T-cell leukemia

Yayoi 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

1Department of Hematology and Oncology, Graduate School of Medicine, Kyoto

University, Kyoto, Japan; 2Department of Endocrinology and Metabolism, Graduate

School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan;

3Department of Internal Medicine, Division of Hematology, Rheumatology and

Respiratory Medicine, Faculty of Medicine, Kagawa University, Miki-cho, Kita-gun,

Japan; 4Division of Cancer Immunology, Exploratory Oncology Research and Clinical

Trial Center (EPOC), National Cancer Center, Kashima, Japan; and 5Department of

Experimental Immunology, World Premier International Immunology Frontier Research

Center, Osaka University, Suita, Japan

*Y.S. and Y.S. contributed equally to this study.

on May 18, 2020. © 2015 American Association for Cancer Research. cancerimmunolres.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 December 17, 2015; DOI: 10.1158/2326-6066.CIR-15-0148

http://cancerimmunolres.aacrjournals.org/

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Running title: Epigenetic alterations of the FOXP3 gene in ATL

Keywords: ATL, FOXP3, TSDR, methylation, HTLV-1

Financial support: This 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.

Corresponding author: Masakatsu Hishizawa

Address: 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan

Tel: 081-(0)75-751-4964

Fax: 081-(0)75-751-4963

e-mail: [email protected]

Disclose of potential conflicts of interest: No potential conflicts of interest are disclosed.

Word counts: 3889

Figure and table counts: 5 and 1




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Abstract

Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell

leukemia virus type 1. Because of its immunosuppressive property and resistance to

treatment, ATL patients have poor prognoses. ATL cells possess the regulatory T cell

(Treg) phenotype such as CD4, CD25, and usually express forkhead box P3 (FOXP3).

However, the mechanisms of FOXP3 expression and its association with Treg-like

characteristics in ATL remain unclear. Selective demethylation of the Treg-specific

demethylated region (TSDR) in the FOXP3 gene leads to stable FOXP3 expression and

defines natural Tregs. Here, we focus on the functional and clinical relationship between

the epigenetic pattern of the TSDR and ATL. Analysis of DNA methylation in

specimens from 26 ATL patients showed that 15 patients (58%) hypomethylated the

TSDR. The FOXP3+ cells were mainly observed in the TSDR-hypomethylated cases.

The TSDR-hypomethylated ATL cells exerted more suppressive function than the

TSDR-methylated ATL cells. Thus, the epigenetic analysis of the FOXP3 gene

identified a distinct subtype with Treg properties in heterogenous ATL. Furthermore, we

observed that the hypomethylation of TSDR was associated with poor outcomes in ATL.

These results suggest that the DNA methylation status of the TSDR is an important

hallmark to define this heterogeneous disease and to predict ATL-patient prognosis.




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Introduction

Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human

T-cell leukemia virus type 1 (HTLV-1) (1, 2). HTLV-1 is endemic in southwest Japan,

Sub-Saharan Africa, the Caribbean Basin, and South America (3). Approximately 5% of

HTLV-1-infected individuals develop ATL after a latency period of 40- to 70-years

from the initial HTLV-1 infection (3, 4). The prognosis of ATL is usually poor with a

median survival period of approximately 1 year (5). The major obstacles in the

treatment of ATL patients include resistance to a variety of cytotoxic agents and

susceptibility to opportunistic infections because of its immunosuppressed property

(3,6,7).

In HTLV-1-infected individuals, HTLV-1 proviral DNA has been detected mainly

in CD4+ T cells (8). However, human CD4+ T cells are heterogeneous, and the subset of

initial HTLV-1 infection in ATL patients remains to be clarified. Most ATL cells express

CD3, CD4, and CD25 (9). This phenotype resembles regulatory T cells (Tregs), and

several previous studies have shown that 60% to 70% of ATL cases expressed forkhead

box P3 (FOXP3) (10, 11), which is a master regulator of Tregs (12).

Tregs play an important role in immune tolerance, and they prevent the

development of autoimmune and inflammatory diseases by suppressing autoreactive T




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cells (13). FOXP3, a key transcription factor, controls both the development of Tregs and

their suppressive function (12–14). However, a substantial amount of evidence indicates

that FOXP3 expression itself is not sufficient to establish the full phenotype and

function of Treg cells (15). In humans, FOXP3+ cells are heterogeneous and are not

always suppressive (16). Tregs are divided into two main subpopulations: natural or

induced Tregs. The former is a genuine Treg, developing in the thymus at the stage of

CD4 single-positive thymocytes (17,18), whereas the latter is induced by differentiation

in the periphery after antigenic stimulation (19,20) and cannot fully exert suppressive

activity (21, 22). Selective DNA hypomethylation of conserved CpG residues within the

FOXP3 locus, termed the Treg-specific demethylated region (TSDR), is important for

distinguishing natural Tregs from peripherally induced Tregs and for stabilizing FOXP3

expression in both humans and mice (23, 24).

Several studies have shown that FOXP3 expression varies among ATL patients

(10, 25), and that FOXP3-expressing ATL cells are not always suppressive (26).

Therefore, we investigated the DNA methylation pattern of the TSDR and its relevance

to Treg properties and clinical outcomes in ATL.




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Materials and methods

Primary ATL cells and control cells

Primary ATL cells were obtained from 40 patients hospitalized at Kyoto

University Hospital or at affiliated hospitals during the period from 2001 to 2014. The

diagnosis of ATL was based on the Shimoyama classification (7). For CpG methylation

analysis, we included only male patients, because one allele of the TSDR on the FOXP3

locus of the X-chromosome was methylated in females (24). The characteristics of 26

male patients are summarized in Table 1 and Supplementary Table S1. Peripheral blood

mononuclear cells (PBMCs) were isolated with Ficoll-Paque (Pharmacia Biotech, Little

Chalfont, UK) density-gradient centrifugation. If the proportion of abnormal cells in the

PBMCs was less than 60%, we purified the ATL cells by sorting (Supplementary Fig.

S1). Control peripheral blood samples were obtained from eight healthy volunteers.

This study was performed in compliance with the Declaration of Helsinki after approval

by the Ethics Committee of Kyoto University, Graduate School of Medicine. Written

informed consent was obtained from all patients and healthy volunteers.

Cell lines

We used HTLV-1-infected cell lines, ATL-43T (27), MT-1 (28), MJ (29), MT-2




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(30), MT-4 (30), and Hut102 (31). ATL43T were kindly provided by Dr M.Maeda

(Virus Institute, Kyoto University, Japan) in 1991. MT-1, MT-2 and MT-4 were obtained

from Japanese Collection of Research Bioresources (JCRB) Cell Bank and Hut102 and

MJ were obtained from American Type Culture Collection (ATCC) between 2000 and

2014. All cell lines were authenticated using HTLV-1 clonal integration and tested for

mycoplasma contamination in 2014. These cell lines were cultured in RPMI-1640

medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum

(Sigma-Aldrich, St. Louis, MO) and 2 mM penicillin, streptomycin, and glutamine

(Gibco BRL, Grand Island, NY) at 37°C in a humidified incubator with 5% CO2 in air.

For the ATL-43T cell line, which is interleukin (IL)2-dependent, the medium was

supplemented with 0.5 nmol/L recombinant IL2 (Shionogi, Osaka, Japan). All cell lines

were not cultured for longer than 1 month.

Bisulfite sequencing

A CpG methylation analysis was performed as described with slight

modifications (23). Briefly, genomic DNA was isolated from cells and bisulfite

treatment was performed using the MethylEasy Xceed Kit (Human Genetic Signatures,

North Ryde, NSW, Australia). Modified DNA was amplified by PCR and cloned into a




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pGEM-T Easy Vector Systems (Promega, Madison, WI). The bisulfite

sequencing-specific primers are listed in Supplementary Table S2. The independent

colonies (16 colonies/region) were amplified with the Illustra TempliPhi Amplification

Kit (GE Healthcare, Buckinghamshire, UK) and sequenced using a 3130xl Genetic

Analyzer (Applied Biosystems, Carlsbad, CA). The percentage of methylation was

calculated by dividing the number of demethylated colonies at the CpG site by 16.

Quantitative reverse transcription-(qRT)-PCR analysis

Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Hilden,

Germany) and converted into cDNA using a SuperScript Reverse Transcriptase

(Invitrogen, Carlsbad, CA) and Random Hexamers (Applied Biosystems). A qRT-PCR

was performed using the TaqMan gene expression kit (Applied Biosystems). The

primers and probes for tax and HTLV-1 bZIP factor (HBZ) were as described (32, 33).

Those for human FOXP3, CTLA4, HELIOS, EOS, β-ACTIN and 18S rRNA were

purchased from Applied Biosystems. We calculated the relative mRNA expression

based on the levels of expression in the MJ cell line. All standards and samples were

analyzed in duplicate, and the average value was used for the calculations.




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Flow cytometric analysis

A phenotypic analysis was performed on the purified PBMCs. The data were

acquired using a FACS LSR II instrument (BD Bioscience, San Diego, CA) or FACS

Aria instrument (BD Bioscience) and analyzed with FlowJo software (ver. 9.7.5; Tree

Star, Inc., San Carlos). For the sorting of primary ATL cells, cells were stained with

anti-CD3 (UCHT1, eBioscience, San Diego, CA), anti-CD4 (RPA-T4, eBioscience),

anti-CD5 (UCH-T2, Biolegend, San Diego, CA), anti-CD7 (CD7-6B7, Biolegend),

anti-CD25 (M-A251, BD Bioscience) and LIVE/DEAD Fixable Dead Cell Stain Kits

(Invitrogen). Cells were sorted with FACS Aria (BD Bioscience) and used for further

experiments (Supplementary Fig. S1).

Functional assay to evaluate the suppressive activity of ATL cells in vitro

The suppressive function of ATL cells was assayed using a modified protocol

(16). Briefly, CD4+CD25−CD45RA+ T cells from a healthy donor were used as

responder cells and stained with the Cell Trace Violet Cell Proliferation Kit (Molecular

Probes, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.

CD4−CD8− cells were used as antigen-presenting cells (APCs) after irradiation (18.5

Gy). Responder cells were cocultured with the same numbers of ATL cells with 10-fold




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APCs, and stimulated with 0.5 μg/mL soluble anti-CD3 (eBioscience) and 0.1 μg/mL

soluble anti-CD28 (eBioscience) in supplemented RPMI medium. The number of

proliferated violet-labeled cells was assessed by flow cytometry after culturing for

84-90 h.

Cytokine staining

Cytokine 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 h in the presence of a Golgi transport inhibitor,

monensin (GolgiStop ; BD Pharmingen, San Diego, CA). After staining with antibodies

to surface moieties, the cells were fixed and permeabilized by Cytofix/Cytoperm and

PermWash (BD Pharmingen) according to the manufacturer’s instructions. Cells were

then stained with anti-cytokine or isotype-matched antibodies and analyzed by flow

cytometry.

Statistical analysis

Statistical analyses were performed using Graph Pad Prism 6.0 (Graph Pad

Software, San Diego, CA). Survival curves of the ATL patients were calculated by the




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Kaplan-Meier method using the SPSS Statistics software program, version 22.0 (IBM,

Armonk, NY). Differences between two groups were evaluated by unpaired t-tests. For

the comparison of multiple groups, we used by Tukey’s method. Comparisons of

overall survival (OS) were carried out between two groups with two-sided log rank tests.

P values < .05 were considered significant.

Results

Methylation status of FOXP3, CTLA4, and HELIOS genes in ATL cells

We first investigated the DNA methylation status of CpG residues in the

FOXP3 gene locus from amplicon (amp) 1 to 11 by using the bisulfite sequencing

method, based on a previous report (24). The MJ cell line and ATL cells from Case 2

showed hypomethylation states of FOXP3 that were similar to those of Tregs, whereas

the MT-1 cell line and ATL cells from Case 17 showed methylation states similar to

those of CD4+CD25− conventional T cells (Fig. 1A). Subsequently, we analyzed the

DNA methylation status of amp 5 of FOXP3 by the same method, because this region,

referred to as the TSDR, is important in the development and function of Tregs (24). We

calculated the mean methylation percentage in the TSDR. A rate lower than 50%,was

considered hypomethylated, and greater than 50%, was considered methylated. ATL




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cells from 15 (58%) out of the 26 patients analyzed have hypomethylation states similar

to normal Tregs (Fig. 1B, left panel). The median methylation rate of 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 methylated in Tregs and

CD4+CD25− conventional T cells (23). We confirmed that the CTLA4 exon 2 and

HELIOS intron 5 regions were hypomethylated in normal Tregs, but highly methylated in

CD4+CD25– conventional T cells (Supplementary Fig. S2). Therefore, we examined the

DNA methylation of both of these areas in primary ATL cells and found that they were

both predominantly hypomethylated, except for four cases in CTLA4 exon 2 and for one

case in HELIOS intron 5. All cases with CTLA4 exon 2-methylation or HELIOS intron

5-methylation were included in the TSDR-methylated cases.

DNA methylation of the TSDR, FOXP3 expression and Treg signatures

We analyzed the expression of Treg-related genes and various cytokines. With the

use of flow cytometric analysis, FOXP3 expression was observed in almost all of the

patients identified with a hypomethylated state. The percentage of FOXP3+ cells was

significantly higher in the TSDR-hypomethylated cases than in the methylated cases




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(Fig. 2A and Supplementary Fig. S3A). The mean fluorescence intensity (MFI) of

FOXP3 tended to be higher in the hypomethylated cases, but the difference was

statistically marginal. Transcriptional analysis by qRT-PCR presented a trend of more

FOXP3 mRNA in the TSDR-hypomethylated cases (Fig. 2B and Supplementary Fig.

S3B). We could not exclude the possibility that contamination of the normal Treg

population might lead to the elevation of FOXP3 expression in whole PBMC samples,

so we isolated the ATL cells by sorting. Purified ATL cells from the

TSDR-hypomethylated cases had significantly more FOXP3 mRNA compared to the

methylated cases (P = 0.01) (Fig. 2B and Supplementary Fig. S3C).

In order to further characterize the ATL cells, we subdivided Tregs into three

fractions (fraction I, II, and III) according to the expressions of CD45RA and FOXP3,

as described (16). We analyzed 8 TSDR-hypomethylated cases and 8 methylated cases.

ATL cells from the TSDR-hypomethylated cases were mainly classified into fraction II

or fraction III, whereas those from the TSDR-methylated cases belonged to various

subsets (Fig. 3). In most of the TSDR-hypomethylated cases,

CD45RA–FOXP3high–activated Tregs constituted a proportion of the ATL cells. ATL cells

from case 8, which had about 40% methylation of the TSDR, also were primarily in

fraction II.




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The expression of other Treg-related proteins besides FOXP3, such as CD25,

CTLA4, GITR, CCR4, and HELIOS, was analyzed in 14 ATL patients, including 8

TSDR-hypomethylated cases (Supplementary Fig. S4). Most patients had expression of

CD25, CTLA4, GITR, CCR4, and HELIOS, consistent with the phenotype of Tregs.

However, the TSDR-hypomethylated and methylated cases were not significantly

different (Supplementary Fig. S4A–E). In addition, expression of proliferation marker

Ki67 (16) was not significantly different between the groups (Supplementary Fig. S4F).

We analyzed cytokine secretion from the ATL cells of 14 patients whose cells were

stimulated with PMA and ionomycin (Supplementary Fig. S5). The cells secreted low

amounts of IFNγ, IL4, IL17, IL10, IL2, and TNFα with no differences between the

TSDR-hypomethylated and methylated cases. Expression of mRNA from CTLA4,

HELIOS, and EOS, which forms a complex with FOXP3 (34), in the cells of 25 ATL

patients varied among the ATL cases, with no significant differences in methylation

status of TSDR (Supplementary Fig. S6). Together, these results indicated that the

common characteristics of Tregs (e.g., FOXP3 expression, cell surface phenotype, and

cytokine secretion) were indicated by hypomethylation of the TSDR in ATL cells.

However, only FOXP3 expression could discriminate between the

TSDR-hypomethylated and methylated cases.




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Functional analysis of suppressive activity of primary ATL cells in vitro

To clarify the functional differences between the TSDR-hypomethylated and

the methylated cases, we measured suppressive activity, a major characteristic of Tregs.

We analyzed 10 ATL cells (5 TSDR-hypomethylated cases and 5 methylated cases).

When we cocultured healthy CD4+CD45RA+ naïve T cells as responders with the

TSDR-hypomethylated ATL cells as effectors, the proliferative activity and number of

responder T cells were suppressed (Fig. 4). In contrast, the TSDR-methylated ATL cells

could not suppress. Similar results were seen in the HTLV-1-infected cell lines

(Supplementary Fig. S7). Thus, the methylation status of the TSDR was associated with

suppressive activity.

HTLV-1 encoded tax and HBZ not related to the hypomethylation of TSDR

It was reported that the HTLV-1–associated molecules tax and HBZ affected

the expression of FOXP3 (35, 36). To clarify the relationship between FOXP3

expression and these molecules, we analyzed mRNA expression.. Consistent with

previous reports (37), ATL cells contained little or no tax mRNA. The methylation

status of the TSDR was not related to the amount of tax mRNA (Supplementary Fig.




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S8A). HBZ mRNA was also independent of the methylation status of the TSDR in ATL

patients (Supplementary Fig. S8B). Taken together, these results show that there is no

obvious relation between HTLV-1 viral molecules and the methylation pattern of the

TSDR in ATL.

Hypomethylation of TSDR as a predictive factor for survival in ATL

To assess the clinical significance of the epigenetic status of FOXP3, we divided

25 patients into two groups according to the TSDR methylation status. One patient

(Case 5) was excluded in this analysis because of missing data. Clinical characteristics

including the median age, clinical type, history of stem cell transplantation (SCT), and

putative prognostic factors such as concentrations of serum albumin, lactate

dehydrogenase (LDH), calcium, IgG, and soluble IL2 receptor not significantly

different between the TSDR-hypomethylated group and the TSDR-methylated group

(Table 1 and Supplementary Table S1). The median OS for the entire group was 315

days (range, 16-3923 days) and 19 of the 25 patients (76%) died. The median OS of the

TSDR-hypomethylated group and that of the TSDR-methylated group were 283 days

(range, 16–3099 days) and 627 days (range, 116–3923 days), respectively

(Supplementary Fig. S9A). As the patients with chronic and smoldering types of ATL




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had better prognoses than those with the acute or lymphoma types, we excluded both of

these clinical types and reanalyzed OS. The TSDR-hypomethylated group had

significantly inferior OS compared to the TSDR-methylated group:

TSDR-hypomethylated, median OS of 186 days (range, 16–691 days),

TSDR-methylated, median of 627 days (range, 116–3923 days), P = .02 (Fig. 5). Of the

ten deceased patients in the TSDR-hypomethylated group, eight patients succumbed to

ATL, one patient to infection, and one to treatment-related mortality. Of the six

deceased patients in the TSDR-methylated group, four patients succumbed to ATL, and

two to infection (Supplementary Table S1). These results suggest that the methylation

pattern of TSDR could be associated with survival for ATL patients.

Next, we analyzed the relationship between the expression of FOXP3 and survival.

Although the high frequency of CD45RA–FOXP3high T cells had a tendency to reduce

survival, this association was not statistically significant (P = 0.14) (Supplementary Fig.

S9B). Even when we include 14 female patients for the analysis, the difference was not

apparent (P = 0.16) (Supplementary Fig. S9C, 10 and Supplementary Table S3).

Thus, the methylation pattern of the TSDR may provide more precise prognostic

stratification than expression of FOXP3.




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Discussion

In this study, we found that ATL cells contain a unique cell subtype with a

Treg-like hypomethylation status in the TSDR. The TSDR-hypomethylated ATL cells

not only had higher expression of FOXP3 and more suppressive activity, but also poorer

clinical outcomes.

FOXP3 expression in ATL cell lines and primary ATL cells has been investigated

by many researchers at the mRNA and protein levels, and it varies widely among cell

lines and individual cases (10, 11, 25, 26, 38, 39). Our data also showed a wide range of

FOXP3 expression among primary ATL cells. Among these heterogeneous clusters, we

could differentially identify one specific subtype, which shows the

TSDR-hypomethylated status similar to that of natural Tregs. These

TSDR-hypomethylated ATL cells resemble natural Tregs not only in their expression of

FOXP3, but also other Treg-specific markers such as CD25, CTLA4, HELIOS, and

GITR. However, the TSDR-methylated ATL cells also expressed Treg-specific markers

other than FOXP3. In conventional T cells, the expression of CD25 and CTLA4 is

readily induced by T cell activation, irrespective of their DNA methylation status (23).

This may explain the expression of such molecules in ATL cells with a methylated

TSDR.




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The suppressive function of ATL cells has also been a subject of considerable

discussion. Some studies show suppressive activity of ATL cells (39, 40), but others did

not (26). We demonstrated that the TSDR-hypomethylated ATL cells, which showed

higher FOXP3 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 is essential for stable FOXP3 expression and

suppressive function (24, 41, 42). Similarly, in ATL cells, we found that the DNA

hypomethylation status of the TSDR was closely associated with FOXP3 expression

and function.

The HTLV-1-encoded genes, tax and HBZ, can modulate the expression of

FOXP3. Transfection of tax DNA to CD4+CD25+ T cells resulted in a reduction of

FOXP3 mRNA and suppressive function (35). In HBZ transgenic mice, the number of

FOXP3+CD4+ T cells increased and these HBZ-induced Tregs had unstable FOXP3

expression, impaired function, and methylated TSDRs (36). However, primary ATL

cells were not assessed in these experiments. In the present study, no correlation was

detected between HTLV-1-associated molecules and the expression of FOXP3 or the

methylation status of the TSDR.

Several studies have tried to identify the origin of ATL cells (11, 43), but the




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answer as to whether the origin of ATL cells is natural Tregs or conventional T cells has

remained controversial. The DNA demethylation of the FOXP3 gene is thought to be a

specific phenomenon of natural Tregs. In addition, the HTLV-1 gene itself could change

FOXP3 expression through the TGFβ signal pathway (44), but such FOXP3+ T cells are

induced Tregs, not natural Tregs. We therefore speculate that the demethylation of the

TSDR in ATL cells is not a secondary event after HTLV-1 infection, and that the ATL

cells with the hypomethylated TSDR may be derived from natural Tregs. However, we

cannot exclude the possibilities that (i) the HTLV-1 virus may have infected

CD4+Foxp3− non-Treg cells, and(ii) epigenetic change had occurred during the

development of ATL. Therefore, we cannot make any conclusions about the origin of

ATL cells from the findings of the present study. More detailed characterizations of the

TSDR-hypomethylated ATL cells at different time points during ATL progression

would provide useful information about the etiology of ATL cells.

ATL is divided into four clinical variants: acute, lymphoma, chronic, and

smoldering. The former two variants have a more aggressive clinical course and shorter

OS than the latter two. The ATL-prognostic index was recently proposed to identify the

clinical risk among acute and lymphoma type patients (45). Until now, the biological

phenotype could not be used to distinguish the prognosis (46).




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With respect to the FOXP3 expression , some studies have evaluated ATL cells by

immunohistochemical staining (25, 47) or qRT-PCR (48), but they did not show a

correlation with OS. Similarly, we couldn’t show a significant association between the

FOXP3 expression and the OS. But the results of the present study demonstrated that

the methylation pattern of the TSDR is correlated with the OS, regardless of the

treatment with SCT (data not shown), and it was maintained when we omitted

TSDR-methylated but FOXP3+ cases (data not shown). The hypomethylation of the

TSDR in ATL cells led to ¥ FOXP3 expression, but even the TSDR-methylated cases

often expressed FOXP3 (Figs. 2 and 3). The hypomethylation of the TSDR in ATL cells

is closely associated with their suppressive function, like natural Tregs. In contrast, the

expression of FOXP3 is not specific to Tregs, and the FOXP3-expressing ATL cells with

the methylated TSDR may be classified with non-Tregs that do not possess suppressive

function. Such functional deference according to the methylation status might influence

the clinical course of ATL. However, the intrinsic immunosuppression in the

TSDR-hypomethylated patients seems to be unrelated to the cause of death in the

present small cohort, and we could not elucidate the reason for their poor survival. The

possibility of other causes, such as drug resistance, will be a subject for future

investigation.




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The limitation of our study was that we could only analyze 26 patient cases of

ATL regarding the methylation pattern of the TSDR, and the treatment strategies

differed among patients. It is necessary to confirm our results by conducting a

large-scale prospective clinical study. In addition, we could not evaluate the methylation

patterns of the TSDR in women. We analyzed other Treg-specific demethylation regions,

e.g., CTLA4 exon 2 and HELIOS intron 5, but the hypomethylation of such regions was

not specific to ATL patients with the hypomethylated TSDR. Therefore, to analyze

female cases, other assessment strategies are needed. In the male transplant setting,

incidence of ATL in Japan is generally higher and its prognosis is worse than in females

(49). We do not know whether our results could be applied to cases involving women.

To date, the definition of hypomethylation is not standardized. We tentatively defined

the cut-off values for FOXP3 DNA hypomethylation as 50%. According to this

definition, we can classify the hypomethylated cases with high FOXP3 expression in

this small cohort, but the relevance of this threshold should be reevaluated with a more

large-scale study. However, we believe that it is important to recognize the unique ATL

subtype defined according to the hypomethylation status of the TSDR. In the future,

different treatment strategies might be needed for the treatment of this poor-prognosis

ATL subtype.




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In conclusion, we propose that a distinct subtype of the heterogeneous ATL

disease can be defined according to the hypomethylated status of the TSDR. This Treg

subtype is related to the biological phenotype and clinical course of ATL. The

hypomethylated status of the TSDR in ATL cells might be a promising tool for risk

classification.

Acknowledgments

The authors are grateful to Dr Maeda (Virus Institute, Kyoto University, Japan)

for providing ATL-43T. We thank clinicians in Japanese Red Cross Wakayama Medical

Center (Wakayama, Japan), Kitano Hospital (Osaka, Japan), Kokura Memorial 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) for providing clinical samples.




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30

Table 1. Patients’ characteristics

Hypomethylated patients Methylated patients P

Age (year), median (range) 57 (33-87) 54 (29-80)

Clinical type

Acute

Lymphoma

Chronic

Smoldering

10 (66.7%)

1 (6.7%)

3 (20.0%)

1 (6.7%)

7 (63.6%)

2 (18.1%)

1 (9.0%)

1 (9.0%)

Alb (g/dl), median (range) 3.9 (2.3-4.4) 3.6 (2.6-4.5) .63

LDH (IU/L), median (range) 1036.5 (323-6451) 485 (195-3092) .46

Ca (mg/dl), median (range) 9.7 (8.8-16.9) 11.2 (9.1-16.9) .38

IgG (mg/dl), median (range) 1093 (236-1395) 990 (624-1460) .80

sIL-2R (U/ml), median (range) 30071 (758-104000) 18670 (1220-232000) .55

OS (days), median (range) 283 (16-3099) 627 (116-3923) .1

Cause of death

ATL

Infection

Others

8 (66.6%)

2 (16.7%)

2 (16.7%)

5 (71.4%)

2 (28.6%)

0

LDH indicates lactate dehydrogenase; sIL-2R, soluble interleukin-2 receptor; and OS,

overall survival.




31

Figure Legends

Figure 1. The Treg-specific demethylated region (TSDR) of ATL cells exhibits

regulatory T cell-like hypomethylated status or CD4+ conventional T cell-like

methylated status.

Bisulfite sequencing was performed using genomic DNA from each sample. The

frequency of the demethylation of CpG is shown. Yellow = hypomethylated and blue =

methylated in each CpG. (A) The DNA methylation status in the TSDR of the

HTLV-1-infected cell lines and primary ATL cells was analyzed, and all TSDR, from

amplicon (amp) 1 to 11, are illustrated. MJ cell line was used as the

TSDR-hypomethylated representative, and MT-1 cell line was used as the

TSDR-methylated representative. For primary ATL cells, Case 2 was used as the

TSDR-hypomethylated representative, and Case 17 was used as the TSDR-methylated

representative. (B) The DNA methylation status of the TSDR of a healthy donor and

primary ATL cells. Regulatory T cells from a healthy donor were used as the

TSDR-hypomethylated control, and CD4+CD25−conventional T cells from a healthy

donor were used as the TSDR-methylated control. The number of primary ATL cells

corresponds to each case number shown in Supplementary Table S1. The data shown

are the sum of three independent experiments.




32

Figure 2. The hypomethylation of the TSDR correlates with FOXP3 expression.

(A) Primary ATL cells were stained with FOXP3 antibody, and the expression level of

FOXP3 was assessed by flow cytometry. Representative histogram data of FOXP3

expression are shown. Case 13 and Case 22 are representatives of the hypomethylated

TSDR (Hypomethylation) and the methylated TSDR (Methylation), respectively. The

frequency of FOXP3-positive cells and mean fluorescence intensity (MFI) of FOXP3

were calculated and plotted. Each dot represents individual ATL cells from each patient,

and bar indicates the median. (B) The FOXP3 mRNA levels of primary ATL cells were

assessed by quantitative RT-PCR method. The FOXP3 mRNA level was divided by the

β-actin mRNA level. The relative mRNA levels were calculated based on the expression

of mRNA level in the MJ cell line. Bars indicate the median. Bold circles and squares

indicate sorted samples. Solid circles and squares indicate unsorted samples. Statistical

analysis was performed and P-value was shown in the figure.

Figure 3. Subpopulation of FOXP3-positive ATL cells.

The expressions of CD45RA and FOXP3 of primary ATL cells are shown. CD45RA:

y-axis, FOXP3: x-axis. We analyzed total 16 cases. 8 TSDR-hypomethylated cases and




33

8 methylated cases). The representative data of a healthy individual is also shown. As

mentioned in a previous study (16), Tregs can be divided into three groups according to

their function: fractions I, II and III. Each square indicates fraction I (Fr I), fraction II

(Fr II) and fraction III (Fr III), respectively. Numbers indicate the frequency of CD4+

Tcells in each fraction.

Figure 4. Primary ATL cells with the hypomethylated TSDR showed suppressive

function resembling regulatory T cells.

CD4+CD45RA+ naïve T cells from a healthy donor were used as responder cells,

cultured with irradiated APC with or without primary ATL cells from patients.

Responder cells and primary ATL cells were added at a 1:1 ratio. ATL cells from Cases

5 and 17 were used as representative TSDR-hypomethylated cases (Hypomethylation)

and TSDR-methylated cases (Methylation), respectively. The proliferation of responder

cells was assessed by the dilution of labeled dye. Representative data of responder cells

alone, ATL cells from the hypomethylated TSDR added, and ATL cells from the

methylated TSDR added (top panel). The numbers indicate the frequency of

proliferating responder cells. The percentage of proliferating responder cells (middle

panel) and the numbers of proliferating cells (bottom panel) were plotted as mean ±




34

standard deviation. Representative data were shown, as the experiments were

independently performed three times using different ATL cells with similar results. P

values were indicated in each figure; NS, not statistically significant.

Figure 5. Overall survival according to the methylation status of the TSDR.

Survival curves of the hypomethylated TSDR (solid line) and the methylated TSDR

(dotted line) were calculated by the Kaplan-Meier method. P value was shown in the

figure.




Figure 1

MJ

MT-1

Case2

Case17

amp1 2 3 4 5 6/7 8 9 10/11

B

A

Healthy donor

CD4+CD25-

conventional T cells

Healthy donor

Regulatory T cells

amp 5 amp 5

1

2

3

4

5

6

7

8

9

10

11

12

13

14

16

17

18

19

20

21

22

23

24

25

26

15

CpG demethylation

0% 100%

Primary ATL cells amp 5 amp 5

MJ

ATL-43T

MT-1

MT-2

MT-4

Hut102

amp 5 HTLV-1-infected

cell lines amp 5




Figure 2

A C

ell

Num

bers

(% M

ax)

100

0

98.2%

FOXP3

Hypomethylation Methylation

% o

f F

OX

P3+

cells

0

20

40

60

80

100

MF

I of

FO

XP

3

Hypomethylation Methylation 0

500

1000

1500

2000

2500

P=.05 Isotype

ATL cells

Hypomethylation

B

Methylation Regulatory T cellls CD4+ naïve T cells

5

10

15

20

FOXP3/β

-AC

TIN

Hypomethylation

0

MFI

=1988

10.3%

Methylation

MFI

=175

FOXP3

Cell

Num

bers

(% M

ax)

100

0

P=.008

P=.079 P=.003





Healthy donor

Fr I: 3.1%

Fr II: 4.3% Fr III: 4.1%

Figure 3

FOXP3

CD

45R

A

1%

3% 14%

Case 26

1%

0.5% 0%

Case 23

5%

51% 28%

Case 10

1.0%

36% 53%

Case 11

0%

82% 16%

Case 12

0.8%

55% 35%

Case 14

0%

25% 0.4%

Case 16

0%

87% 0.3%

Case 17

0.2%

64% 31%

Case 18

0.2%

7.5% 2.8%

Case 22

0%

72% 0.4%

Case 25

0.4%

93% 4%

Case 13

0%

84% 7%

Case 4

0%

70%

Case 5

20%

Case 24

3%

40% 6%

0%

34% 63%

Case 8




Figure 4

0

20

40

60

80

100

87.5% 64.2% 82.6%

Responder

alone


Hypomethylation Methylation Responder

alone

% o

f pro

lifera

tive c

ells

Responder cells : ATL cells

= 1 : 1

Tota

l num

be

rs o

f re

spon

der

cells

Cell

Num

bers

(% M

ax)

100

0 Violet dye

58.7%

Responder+

Treg

0

10000

20000

30000

40000

50000

60000

70000

Responder

+Treg

Hypomethylation Methylation Responder

alone

Responder

+Treg

P=.01 P=.01

NS

P=.02

P=.01 P=.06

NS

P=.01

NS

P=.03

NS

P=.07




Figure 5

Days after diagnosis (days)

Overa

ll surv

ival

Methylation

Hypomethylation

1.0

0.8

0.6

0.4

0.2

0

P=.02

0 1000 2000 3000 4000




Published OnlineFirst December 17, 2015.Cancer Immunol Res Yayoi Shimazu, Yutaka Shimazu, Masakatsu Hishizawa, et al. T-cell leukemiaFOXP3 is a hallmark of the regulatory T-cell subtype in adult Hypomethylation of the Treg-specific demethylated region in

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