Human natural Treg microRNA signature: Role of microRNA‐31 and microRNA‐21 in FOXP3 expression

Human natural Treg microRNA signature: Role ofmicroRNA-31 and microRNA-21 in FOXP3 expression

Redouane Rouas�1, Hussein Fayyad-Kazan�1, Nabil El Zein1,

Philippe Lewalle1, Franc-oise Rothe2, Alexandru Simion3, Haidar Akl1,

Mohamad Mourtada4, Mohamad El Rifai1, Arsene Burny1,

Pedro Romero5, Philippe Martiat��1 and Bassam Badran��1

1 Laboratory of Experimental Hematology, Institut Jules Bordet, Universite Libre de Bruxelles,

Bruxelles, Belgium2 Translational Research Unit, Institut Jules Bordet, Universite Libre de Bruxelles, Bruxelles,

Belgium3 Universite Catholique de Louvain and de Duve Institute, Brussels, Belgium4 Department of Biochemistry, Laboratory of Immunology, Faculty of Sciences, Lebanese

University, Hadath Beirut, Lebanon5 Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne

Branch, Lausanne, Switzerland

Treg are the main mediators of dominant tolerance. Their mechanisms of action and

applications are subjects of considerable debate currently. However, a human microRNA

(miR) Treg signature has not been described yet. We investigated human natural Treg and

identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two

were considerably under-expressed (miR-31 and miR-125a). We identified a functional

target sequence for miR-31 in the 30 untranslated region (30 UTR) of FOXP3 mRNA. Using

lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-

21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates

FOXP3 expression by binding directly to its potential target site in the 30 UTR of FOXP3

mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator

of FOXP3 expression. Transduction of the remaining three miR had no direct effect on

FOXP3 expression or on the phenotype and will remain the subject of future investiga-

tions. In conclusion, not only have we identified and validated a miR signature for human

natural Treg, but also unveiled some of the mechanisms by which this signature was

related to the control of FOXP3 expression in these cells.

Key words: Human . MicroRNA . Treg

Introduction

It has been established that Treg are the main mediators of dominant

tolerance [1, 2]. Their mechanisms of action as well as their potential

applications are subjects of considerable debate at the moment.

Although a variety of T lymphocytes with suppressive functions have

been described, mainly two subsets of CD41 Treg appear to be better

characterized, i.e. the thymus-derived natural Treg (nTreg) and the

peripherally induced Treg, which derive from naıve T-cell precursors.

nTreg are characterized by the expression of the fork head-winged

helix transcription factor FOXP3 and of the a chain of IL-2 receptor

(CD25). Naturally arising CD41CD251 Treg can inhibit or alter most

�These authors contributed equally to this work.��These authors are joint senior authors.

Correspondence: Dr. Philippe Martiate-mail: [email protected]

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200838509 Eur. J. Immunol. 2009. 39: 1608–1618Rouas Redouane et al.1608

types of immune responses, either innate or acquired [3]. Consider-

ing their immunoregulatory characteristics, Treg represent a very

attractive cell population in the context of immunotherapy of cancers,

immune disorders, infectious diseases and in the field of post-

transplantation immunomodulation. Thus, understanding their

biology as well as developing tools for their accurate identification

and manipulation is central for discovering novel immunotherapeutic

strategies.

FOXP3 has generally been described as a master regulator of

Treg development and function [4–6], both because its expression

in T cells generates an nTreg-like phenotype and function, and

because its absence or inactivating mutations are at the basis of

multiple immune disorders with often fatal consequences [7, 8].

Human FOXP3 has also been involved in nTreg function. The

immune dysregulation polyendocrinopathy and enteropathy

X-linked syndrome, a lethal inherited lymphoproliferative disease

in children has been linked to mutations in the FOXP3 gene.

However, in contrast to the pattern of FOXP3 expression in

experimental mice, human FOXP3 has been shown to be a less

stringent Treg marker. Indeed, FOXP3 is transiently expressed

after T-cell activation [9–12]. Additional phenotypic markers

such as low cell-surface expression of the IL-7Ra chain: CD127

[13] or epigenetic traits [14] may be needed to precisely define

FOXP31 T cells as bona fide nTreg.

Human cord blood contains a distinct population of CD251

Treg [15–17], less heterogeneous than in adult peripheral blood.

Indeed, unless fetal infection, the majority of cord blood CD41

CD251 T cells express FOXP3. Thus, cord blood is a convenient

source for studies aiming at understanding human nTreg

because, in contrast to adult blood, they are less contaminated by

CD251-activated non-Treg T cells. Moreover, while FOXP3

expression is not sufficient to identify Treg in adult human blood,

it remains a marker of choice in umbilical cord blood (UCB)

lymphocytes from healthy newborns and in adult peripheral

blood Treg provided they are distinguished from activated T cells.

MicroRNA (miR) are small single-stranded non-coding RNA

molecules of about 21–23 nucleotides in length, which regulate gene

expression by targeting mRNA for degradation or translational

repression at the post-transcriptional level. Studies in human cells

have only begun to reveal the mechanisms of miR regulation activity,

the normal physiological functions they influence and how their

alteration can lead to pathologies [18]. miR recognize their targets by

base pairing with a short region of about seven nucleotides, either in

the translated region, or in the 30 untranslated region (30 UTR) of

their target mature mRNA. On recognition by RNA Induced Silencing

Complex, target mRNA translation is inhibited or, otherwise, the

target itself is committed to degradation.

An miR signature has been described in mouse Treg, but not yet

in their human counterpart [19]. We therefore investigated the miR

expression profile of human nTreg, purified from cord blood cells, in

comparison with their non-Treg counterpart. We identified a signa-

ture consisting of five miR, the expression of which was statistically

different from the non-Treg CD41 T cells. Among those, miR-31 and

miR-125a were markedly under-expressed. Since the down-regulated

miR can be assumed to result in the up-regulation of the expression of

its target genes, we next screened several genes sequences for

potential target sites for these miR. As far as FOXP3 was concerned,

we were able to identify a potential miR-31 target sequence in the

30 UTR of FOXP3 mRNA, but not for the other four miR of the Treg

signature. Using several types of experiments, including lentiviral

transduction of fresh T cells, we found evidence for a major direct

role of miR-31 in the regulation of FOXP3 expression. We also found

that FOXP3 expression was positively regulated by miR-21. In this

paper, we defined for the first time a human nTreg miR signature and

demonstrated the major direct role played by miR-31 in the regula-

tion of FOXP3 as well as an indirect one for miR-21.

Results

Human nTreg miR signature

Human naturally occurring Treg and CD41CD25� T cells subsets

were isolated using immunomagnetic selection from a series of

Figure 1. Representative flow cytometry plots of purified human UCBCD41 T cells stained for FOXP3. Intracellular FOXP3 expression wasdetermined by flow cytometry in gated CD41 T cells to exclude celldebris. Histograms illustrate FOXP3 expression (filled histograms) withrespective isotype control (empty histograms) in total CD41 T cells. Thecells were sorted by immunomagnetic cell sorting and FOXP3 expres-sion was analyzed in CD41CD25� and CD41CD251 T-cell fractions.Results are representative of five independent experiments.

Eur. J. Immunol. 2009. 39: 1608–1618 Molecular immunology 1609


UCB lymphocyte samples. Flow cytometry after intracellular

FOXP3 staining was performed on each sample to confirm protein

expression in the CD41CD251 fraction and its absence in the

CD41CD25� fraction (Fig. 1). RNA was extracted from pairs of

nTreg and their CD41CD25� T-cell counterparts isolated from

five independent UCB samples. Each sample pair was studied

using TaqMan Low Density Array (TLDA). The data sets were

subjected to significant analysis of miR, in which each miR was

assigned a score on the basis of its change in expression relative

to the standard deviation of repeated measurements. We

identified ten miR that were differentially expressed between

nTreg and naıve CD41CD25� T cells (Table 1).

Eight miR were preferentially expressed in nTreg (miR-425-

5p, miR-181c, miR-21, miR-374, miR-586, miR-340, miR-26b

and miR-491) and two were selectively down-regulated in nTreg

(miR-31 and miR-125a) using the TLDA technique. Differential

miR expression was then validated by real-time PCR (Fig. 2). An

nTreg miR signature was defined consisting of five statistically

differentially expressed miR (21, 31, 125a, 181c and 374). These

miR were detected using the TLDA technique and confirmed by

quantitative PCR. Three of the five miR were over-expressed (21,

181c and 374) and the remaining two were under-expressed (31

and 125a).

Lentiviral transduction of regulatory and non-regulatory UCB T-cells

Lentiviral-based systems have been developed to generate

replication incompetent lentiviral vectors that efficiently trans-

duce both dividing and non-dividing mammalian cells and

provide stable, long-term expression of RNA of interest [20,

21]. To investigate the effect of some miR of this signature

(miR-31, miR-21) on Treg and CD41CD25� T lymphocytes

and more specifically on FOXP3 expression, lentiviral vectors

(lenti-miR-31, lenti-miR-21, lenti-miR-125a) containing copepod

GFP as a reporter gene were produced and transduced into cord

blood Treg and non-Treg after 18 h stimulation period with IL-2

and PHA. To rule out any artifact due to the stimulation, we

verified that the signature, i.e. the ratio for the five miR found in

Treg versus non-Treg did not change upon activation. We found

that activation, prior to transduction, augmented some miR

expression (especially miR-21 and mir-31) in both types of cells,

but by the same factor (approximately threefold) as presented in

Table 2. In addition, the Treg/non-Treg ratio of FOXP3

expression levels remained the same in activated and non-

activated T cells. A scrambled lenti-miR-ctrl was used as a

negative control. After optimization of transduction conditions

Table 1. nTreg miR signature using the TLDA techniquea)

miRNA Treg/CD41CD25� ratio p-Value

miR-491 2.3 0.001

miR-21 2.7 0.01

miR-425-5p 2.1 0.01

miR-31 0.2 0.03

miR-586 4.7 0.02

miR-181c 1.8 0.02

miR-340 2.6 0.03

miR-26b 2.5 0.03

miR-125a 0.6 0.04

miR-374 2.9 0.04

a) The TLDA technique identified ten miR differentially expressedbetween CD41CD251 T cells (nTreg) and the CD41CD25� fraction,with a p-value o0.05. The expression of each of these ten miR wasthen subjected to an individual quantitative PCR assay.

Figure 2. Five miRNA are significantly differentially expressed between CD41CD25� and CD41CD251 T cells from five independent donors. Dataobtained by quantitative RT-PCR amplification of miR are plotted. p-Values for each miRNA are shown. Boxes represent SE. Error bars represent SD;pooled data from five independent experiments. �po0.05, ��po0.01 CD41CD251 versus CD41CD25� T cells (Student’s t-test).

Eur. J. Immunol. 2009. 39: 1608–1618Rouas Redouane et al.1610


using adult CD41 T cells, we defined an MOI of five as the one

providing the highest transduction rate. This MOI was then

chosen to transduce purified CD41CD25� and CD41CD251 UCB

T cells. The efficacy of transduction was measured 1 wk after

transduction. Measurement of GFP expression by flow cytometry

showed a 75–87 and 70–85% transduction efficiency of,

respectively, lenti-mir-31 and lenti-miR-21. Subsequent flow

cytometry sorting of the GFP1 cells resulted in a 99% pure

population (Fig. 3).

Lentiviral expression of miR-21 in non-Treg T cellssignificantly increases the FOXP3 level

After GFP1 cell sorting, FOXP3 mRNA and miR-21 expression

were assessed after transduction by lenti-miR-21 and lenti-miR-

ctrl, in non-Treg. Our results showed that miR-21 level in lenti-

miR-21 transduced non-Treg was threefold higher than in lenti-

miR-ctrl transduced T cells (Fig. 4). At the same time the level of

FOXP3 expression was twofold higher in lenti-miR-21 transduced

lymphocytes compared with lenti-miR-ctrl and non-transduced

cells. Furthermore, FOXP3 protein level was checked after lenti-

miR-21 transduction. As shown in Fig. 5B FOXP3 protein level

increased significantly after lenti-mir-21 transduction compared

with lenti-miR-Ctrl transduced CD41CD25� T cells. These

results reveal that FOXP3 expression is positively regulated by

miR-21.

Lentiviral expression of miR-31 in nTreg significantlyreduces FOXP3 transcription and protein level

In order to study the effect of over-expression of miR-31 on Treg

and specifically on FOXP3, lenti-miR-31 was transduced into Treg

and the GFP1 cells were sorted. Figure 5A shows that miR-31

level in lenti-miR-31 transduced Treg was significantly higher

than in lenti-miR-ctrl transduced cells while the level of miR-31

was threefold lower in Treg compared with their negative

counterpart. Obviously, the level of expression of miR-31 in

transduced Treg was higher than the physiological level observed

in non-Treg cells, but this is due to the impossibility of adjusting

the level of expression of the transgene. It nevertheless shows

that miR-31 is able to down-regulate FOXP3 expression, albeit

not to the level of non-Treg, demonstrating that FOXP3

expression must be controlled by the combination of several

pathways, including miR-21 and miR-31. In addition, the level of

FOXP3 was twofold lower in lenti-miR-31 transduced cells

compared with lenti-miR-ctrl and non-transduced cells. We next

examined FOXP3 protein level after lenti-miR-31 and lenti-miR-

ctrl transduction. Figure 5B shows that FOXP3 protein level was

significantly reduced in lenti-miR-31 transduced cells compared

with lenti-miR-ctrl and non-transduced cells. In contrast,

transduction of lenti-miR-125a in Treg had no effect on FOXP3

expression compared with the lenti-miR-Ctrl transduced Treg.

Table 2. Changes upon activation of miR-31 and miR-21 expression innTreg and non-Treg by quantitative PCRa)

Fold change upon activation

CD41CD25� nTreg

miR-21 3.25 3.03

miR-31 2.59 2.34

a) To achieve an optimal transduction level, T lymphocytes had to beactivated, which can modify some miR expression. Each miRexpression was evaluated by quantitative PCR before and afteractivation. Two (miR-21 and miR-31) were found to be up-regulated,but in the same proportion, meaning that the signature, defined asan miR differential expression between CD41CD251 and CD41CD25�

remained the same.

Figure 3. Lentiviral transduction efficiency in nTreg. GFP expression inTreg was assessed 1 wk after transduction with lentiviral vectorharboring miR-31 before and after fluorescence-activated cell sorting.Data shown are representative of three independent experiments.

Figure 4. Lentiviral-mediated miR-21 expression and its effect onFOXP3. MiR-21 and FOXP3 relative expression in untransduced non-Treg (CD41CD25�), miR-21 or miR-Ctrl transduced CD41CD25� T cells,and in untransduced Treg (CD41CD251) as determined by miR-quantitative RT-PCR. Data represent mean7SD of three independentexperiments, each performed in triplicate. �po0.05 (lenti-miR-21 versuslenti-miR-ctrl transduced non-Treg; Student’s t-test).



These results demonstrate that FOXP3 expression is inversely

proportional to miR-31 expression level.

FOXP3 is directly regulated by miR-31

Since FOXP3 is the hallmark of nTreg and is known to be

mandatory for nTreg cell function [4, 5], we then decided to

investigate whether miR-31 might influence the expression of this

gene through its 30 UTR target sequence. Thus, we cloned a

300 bp fragment of the 30 UTR of FOXP3 that contains the mir-31

target sequence (Fig. 6) in a psiCHECK-1 vector downstream of

the Renilla luciferase gene (psiCHECK-UTRwt). In parallel, the

conserved target site of miR-31 was deleted (psiCHECK-UTRdel)

by site-directed mutagenesis. Transient transfection of psiCHECK-

UTRwt in HeLa cells led to a significant decrease (65%) in

reporter luciferase activity compared with the control vector

psiCHECK (Fig. 6A). As expected, the activity of the reporter gene

carrying the deletion in the miR-31 target site was the same as

that of the control vector (Fig. 6A). The same observation was

made in 293T cells (Fig. 6B). Furthermore, co-transfection of

HeLa cells with psiCHECK-UTRwt and miR-31 showed a

significant decrease (48%) in reporter luciferase activity

compared with psiCHECK-UTRwt alone (Fig. 6A), while

psiCHECK-UTRwt activity was not affected by co-transfection

with miR-Ctrl. Moreover, the activity of the psiCHECK-UTRdel

deleted at the miR-31 target sequence was not affected by co-

transfection with miR-31 (Fig. 6A).

Anti-miR-31 transfection leads to increased FOXP3expression

In an effort to confirm that miR-31 suppresses directly the

expression of FOXP3 by targeting its 30 UTR, we have assessed the

effect of anti-miR-31 treatment on FOXP3 expression. Since HeLa

cells weakly expressed FOXP3 and expressed miR-31 at a level

similar to non-Treg cells, we transfected them with either a

control-anti-miR or anti-miR-31. Real-time quantitative RT-PCR

was performed 24 h post transfection. At this time point, the

expression of FOXP3 increased 14-fold in cells transfected with

anti-miR-31 compared with mock treated cells or control-anti-

miR transfected cells (Fig. 7). Thus, this observation further

emphasizes the role of miR-31 in controlling the expression of

FOXP3.

Discussion

In this report we define a human nTreg miR signature. To our

knowledge, this is the first human nTreg miR signature to be

characterized. It consists of five miR (21, 31, 125a, 181c and 374)

differentially expressed in UCB CD41CD25� and CD41CD251

T-cell populations. These miR were detected using the TLDA

technique and confirmed by quantitative PCR. Three of the five

miR were over-expressed (21, 181c and 374) and the remaining

two were under-expressed (31 and 125a). Some of them, miR-21

and miR-125a, were identical to those found in the mouse Treg

miR signature and therefore deserved to be thoroughly investi-

gated. In contrast, miR-181c, miR-31 and miR-374 were specific

to human nTreg when compared with the mouse Treg signature.

It is important to emphasize that we describe the miR signature

corresponding to ex vivo non-activated nTreg, which therefore

does not necessarily reflect the adult Treg signature (the

proportion of naıve versus non-naıve cells could be very

different). Future work will be needed to explore the induced

adult Treg miR signature. This will allow a fair comparison with

the mouse signature, derived from a similar population.

Figure 5. (A) Lentiviral-mediated miR-31-expression and its effect onFOXP3 in T cells. miR-31 and FOXP3 expression determined by miR-quantitative RT-PCR in Treg, miR-31 or miR-Ctrl transduced Treg, andin non-Treg. Mean of three independent experiments are shown.(B) Western blot analysis of FOXP3 protein level after lenti-miR-31,lenti-miR-21 and lenti-miR-125a transduction. Cells were collected 7days after transduction. Data represent mean7SD of three indepen-dent experiments, each performed in triplicate. �po0.05, ��po0.01 (lenti-miR-31 versus lenti-miR-Ctrl transduced Treg; Student’s t-test).



Except for over-expression of miR-21, which is not strictly

specific to Treg, although, in any circumstances, its expression

remains higher than in other T cells types, this signature is unique

to natural naıve Treg among all T-cells. The role of these five miR

in human nTreg was largely unexplored. To gain further insight

into the functional role of this signature, we chose to explore the

role of the five miR composing the signature: miR-21, 181c and

374 (over-expressed), and miR-31 and miR-125a, which are

markedly under-expressed in nTreg and therefore should be

mirrored by up-regulated expression of their target genes. The

read-out was FOXP3 expression and protein level. Only two miR

(21 and 31) had an observable effect on these parameters. After

computer screening, we focused our analysis mainly on miR-31

because of its potential binding site in the 30 UTR of FOXP3.

It should also be noted that activation is still required to achieve

a highly efficient lentiviral transduction of T cells and to keep them

alive in culture. Since activation of human T cells transiently induces

FOXP3 up-regulation [9–12], it was necessary to verify the impact of

stimulation on the expression of both the miR signature and FOXP3.

The signature, i.e. the ratios of the five miR between the CD41CD251and CD41CD25� populations, did not change, despite a two- to

threefold increase in miR-21 and miR-31 expression, in both

populations, upon activation. The same was true for the level of

FOXP3 transcripts: although there was a slight increase in both

populations (roughly twofold), the ratio remained the same. This

observation could provide an explanation for the transient increase

in FOXP3 expression in activated T cells, via the activation-induced

up-regulation of miR-21 upon stimulation. A critical question may

arise concerning the impact of miR-31 at physiological levels on

FOXP3 expression in conventional T cells. It is to be noted that we

first tried several ways of transfecting these T cells to get a more

physiological level of miR-31 expression (electroporation, lipofec-

tion, dextran) and none gave us a reasonable percentage of trans-

fected cells (always less than 10% and on average 5%). Only

lentiviral transduction was efficient enough to achieve, on average,

70% transduction efficiency, but the downside of this method is that

it is highly difficult to regulate the expression in order to achieve a

physiological level after transduction. Nevertheless, several authors

(reviewed in [22]) have shown that a 50% reduction of the target

mRNA seemed to be the maximum achievable, even using a

combination of several miR.

Figure 6. Renilla luciferase reporter assays with constructs holding FOXP3 30 UTR sequences from the indicated genes were cotransfected intoHeLa (A) or 293T (B) cells along with a firefly luciferase transfection control plasmid either alone or together with miR-31. Shown are relativeluciferase values normalized to transfections without miRNA. Data represent mean7SD of three independent experiments, each performed intriplicate. �po0.05 versus psiCHECK transfected cells; Student’s t-test.

Figure 7. MiR-31 inhibition increases FOXP3 expression. HeLa cellstransfected with anti-miR-31 showed more than 14-fold increase inFOXP3 expression. Data represent the mean7SD of five independentexperiments, each performed in triplicate. ��po0.01 versus anti-miR-ctrl transfected cells; Student’s t-test.



First, our results demonstrate that miR-31 is a negative

regulator of FOXP3 expression in fresh nTreg. Moreover, its

impact is directly linked to the target site found in the 30 UTR of

the FOXP3 gene, as evidenced by our transfection experiments.

Next, we found miR-21 to positively regulate FOXP3 expression.

Since it is over-expressed in the Treg population and since

no potential target sequence in FOXP3 mRNA could be

identified, the mechanism of FOXP3 up-regulation may be

indirect. The study of the role of miR-125a remains crucial, both

because it is under-expressed in Treg, suggesting that it should

lead to up-regulated expression of other proteins central to Treg

function, and because it is one of the two miR of the signature

conserved across species. This will be the subject of future work,

especially since the five miR from the signature have target sites

in several genes that could be critical for immunoregulatory

functions.

Future work should also address some mechanistic questions such

as the precise role of miR-31 in Treg: is it a major player (apart from

miR-21) in the regulation of FOXP3 expression or does it synergize

with other levels of transcriptional control, genetic or epigenetic?

What mechanisms are responsible for the specific regulation of the

five miR found in the signature? How does miR-21 indirectly regulate

FOXP3 expression? And finally what is the role of the other three miR

found in the signature with regard to the regulatory function of these

cells? Computer analysis showed that the 30 UTR of the following

genes had at least one potential miR target site: FOXP3 (miR-31),

CTLA-4 (miR-181c), IL-10 (miR-374), IL-2 (miR-181c), CD28 (miR-

31), IFN-g (miR-125a and miR-181c), CD25 (miR-21), TGF-b (miR-

425-5p), histone deacetylase (HDAC) 1 (miR-31 and miR-125a),

HDAC2 (miR-21 and miR-31) and HDAC9 (miR-21, miR-31, and

miR-181c) and others containing some of these miR differentially

expressed in Treg. These possibilities will need to be addressed in

future works, using micro-array analyses of transduced T cells.

Indeed, a genomic analysis of FOXP3-transduced cells suggested that

some elements of the Treg gene expression profile might be inde-

pendent of this transcription factor [23, 24]. The transduction of

naıve CD41CD25� T lymphocytes with a FOXP3-encoding retroviral

vector resulted in significant FOXP3 expression. However, this was

not sufficient to induce neither suppressive phenotype nor up-regu-

lation of Treg surface markers [25]. Moreover, Cobb et al. [19]

showed, in mice, that ectopic FOXP3 expression, after CD41 T-cell

transduction, could lead to the expression of some of the murine Treg

miR signatures. Those last observations demonstrate that FOXP3 is

not sufficient for inducing Treg function even though it participates,

directly or indirectly, in mice Treg miR expression profile.

nTreg represent a good model to understand immune toler-

ance to self and non-self antigens. In this regard our data may

contribute to advance the understanding of the underlying

mechanisms modulating Treg function.

In summary, we have not only found and validated a miR

signature for human nTreg but also unveiled some of the

mechanisms by which this signature may be involved in the

control of the FOXP3 levels in nTreg. This opens several possi-

bilities for the future, i.e. to qualitatively investigate induced Treg

in several circumstances, healthy volunteers and patients suffer-

ing from cancer and immune disorders. These experiments,

coupled with quantitative measurements in these different

situations, could provide new insights into the defective immune

response in malignancies, immune disorders, infections and

transplantation.

Material and methods

Collection and preparation of UCB samples

After informed consent, umbilical cord blood mononuclear cells

(UCBMC) were derived from the umbilical vein after normal full-

term deliveries. UCBMC were isolated by centrifugation over a

Ficoll-Hypaque gradient (PAA laboratories).

Isolation of T-cell populations

Cord blood CD41 lymphocytes were purified using CD41 T-cells

isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)

according to the manufacturer protocol. Briefly, UCBMC were

first incubated in PBS supplemented with 2% decomplemented

fetal bovine serum and saturating amounts of biotin-conjugated

antibody cocktail (CD8, CD14, CD16, CD19, CD36, CD56,

CD123, TCR-g/d and Glycophorin A). Cells were then incubated

with antibiotin micro-beads and CD41 cells were purified using

magnetic separation columns (Miltenyi Biotec). The negatively

selected cells were incubated with anti-CD25 micro-beads, then

CD251 and CD25� cells were purified from CD41 T-cell fraction

using magnetic columns (Miltenyi Biotec).

FOXP3 intracellular staining

FOXP3 intracellular staining was performed using Alexa Fluors

647 anti-human FOXP3 Flow Kit (BioLegend, San Diego, CA,

USA) following the manufacturer’s instructions. Flow cytometry

analysis was performed on a FACSCalibur machine with Cell

Quest software (Becton-Dickinson Biosciences).

Cell culture

The HeLa cell line was cultured in DMEM (Lonza, Verviers,

Belgium) supplemented with 10% heat inactivated fetal bovine

serum (Invitrogen Europe, Paisley, UK), 2 mM L-glutamine,

50 IU/mL Penicillin and 50 mg/mL Streptomycin (all from Lonza).

RNA extraction, RT and real-time PCR quantification

Total RNA was extracted from cells using Trizols total RNA

isolation reagent (Roche Diagnostics, Belgium). The concentra-



tion was quantified using a NanoDrop Spectrophotometer.

TaqMan miR assays (Applied Biosystems) were used to quantify

mature miR expression. RNU44 (Applied Biosystems) was used as

endogenous control for miR expression studies.

Thus, gene-specific RT was performed for each miR using

10 ng of purified total RNA, 100 mM dNTP, 50 U MutliScribe

reverse transcriptase, 20 U RNase inhibitor, and 50 nM of gene-

specific RT primer samples using the TaqMan MicroRNA Reverse

Transcription kit (Applied Biosystems). Fifteen microliter reac-

tions were incubated for 30 min at 161C, 30 min at 421C, and

5 min at 851C to inactivate the reverse transcriptase. Real-time

RT-PCR (5 mL of RT product, 10 mL TaqMan 2�Universal PCR

master Mix, (Applied Biosystems) and 1 mL TaqMan MicroRNA

Assay Mix containing PCR primers and TaqMan probes) were run

in triplicates at 951C for 10 min followed by 40 cycles at 951C for

15 s and 601C for 1 min.

Quantitative miR expression data were acquired and analyzed

using an ABI Prism 7900HT Sequence Detection System (Applied

Biosystems).

TLDA and individual quantitative PCR validation

The TLDA (Applied Biosystems) technique allowed us to measure

the expression of 384 miR, in order to compare the miR

expression profile of purified nTreg with their CD25� counterpart

T cells originating from five different cord bloods. TLDA loaded

with TaqMans Gene Expression Assays (Applied Biosystems)

were used for quantitative RT-PCR analysis of selected genes.

cDNA was synthesized from 100 ng of total cellular RNA by First

Strand cDNA Synthesis System using 100 mM dNTP, 50 U

MutliScribe reverse transcriptase, 20 U RNase inhibitor, 10�RT buffer, and Multiplex RT human primer. Ten microliter

reactions were incubated for 30 min at 161C, 30 min at 421C,

5 min at 851C to inactivate the reverse transcriptase.

Five microliter cDNA were mixed with 307mL nuclease free

water and 50 mL of this diluted cDNA were mixed with 50 mL of

TaqMan Universal PCR Master Mix (Applied Biosystems). After

that, 100 mL of the sample-specific PCR mixture was loaded into

one sample port, the cards were centrifuged twice for 1 min at

280g and sealed to prevent well-to-well contamination. The cards

were placed in the Micro Fluidic Card Sample Block of

an ABI Prism 7900 HT Sequence Detection System (Applied

Biosystems). The thermal cycling conditions were 2 min at 501C

and 10 min at 951C, followed by 40 cycles of 15 s at 951C and

1 min at 601C.

In each sample, average cycle threshold (Ct) values for the

target genes were subtracted from the average Ct value of the

reference gene to yield the DCt value. 2-DCt values were calcu-

lated to indicate the relative amount of transcripts in each

sample.

For each miR that was found differentially expressed,

a validation by individual quantitative PCR was performed,

and we retained only the miR concordant with the two

techniques.

MiR-31 target prediction

Computer-based programs were used to predict potential miR-31

targets. Using ‘‘hsa-miR-31’’ as a search term, we searched

miRBase [26] (http://microrna.sanger.ac.uk/), and TargetScan

4.2 [27] (http://www.targetscan.org/).

FOXP3 was identified by both programs as a putative miR-31

target gene. Mir-31 potential target sequence in FOXP3 30 UTR is

presented in Table 3.

Table 3. Results of the search for a miR-31 target site in FOXP3 30 UTRa)

FOXP3 3' UTR cctcaa gatcaaggaa

1501 aggaggatgg acgaacaggg gccaaactgg tgggaggcag aggtggtggg ggcagggatgTarget miR-31

1561 ataggccctg gatgtgccca cagggaccaa gaagtgaggt ttccactgtc ttgcctgcca 1621 gggcccctgt tcccccgctg gcagccaccc cctcccccat catatccttt gccccaaggc 1681 tgctcagagg ggccccggtc ctggccccag cccccacctc cgccccagac acacccccca 1741 gtcgagccct gcagccaaac agagccttca caaccagcca cacagagcct gcctcagctg 1801 ctcgcacaga ttacttcagg gctggaaaag tcacacagac acacaaaatg tcacaatcct 1861 gtccctcac

Target site base pairing

5'.GUGAGGUUUCCACUGUCUUGCCU-3’ Position 125-131 of FOXP3 3’UTR ||| |||||||

3' .GUCGAUACGGUCGUAGAACGGA-5’ hsa-miR-31

a) The search has been performed using two different concordant algorithms (miRBase and TargetScan).



Plasmids construction

A 300 bp fragment encompassing the entire FOXP3 30 UTR was

cloned downstream of the Renilla luciferase gene (EcoRI/XhoI

sites) in the psiCheck-1 plasmid (Promega) and designated as

psiCHECK 30 UTR WT. PCR primers used for amplification of the

FOXP3 30 UTR were:

Forward primer: 50-GCGCCTCGAGGACCTCAAGATCAAGG-

AAAGGA-30

Reverse primer: 50-GCGCGAATTCTGAGGGACAGGATTGTG-

ACAT-30

QuikChange site-directed mutagenesis (deletion) of miR-31

target site in psiCHECK 30 UTR WT was performed according to

the manufacturer’s protocols (Stratagene) and designated as

psiCHECK-UTRdel. QuikChange site-directed mutagenesis was

performed using the following primers:

Forward primer: 50-GGTTTCCACTGGCCAGGGCCCC-30

Reverse primer: 50-GGGGCCCTGGCCAGTGGAAACG-30

The constructs were verified by sequencing.

Luciferase assays

Assays were conducted in a 24-well format. Reporter plasmids

(psiCHECK/psiCHECK 30 UTR WT/psiCHECK 30 UTR deleted)

(100 ng) were co-transfected into HeLa cells along with miR-31-

mimic/miR-negative control-mimic at final concentration of

10 mM (miRIDIAN mimic, Dharmacon) and control firefly

plasmid pGL3-CMV (100 ng) using Lipofectamine 2000

(Invitrogen) according to the manufacturer’s guidelines.

Twenty-four hours post transfection, cells were harvested

and luciferase levels were measured using the Dual luciferase

reporter assay system (Promega) according to the manufacturer’s

guidelines. Relative protein levels were expressed as Renilla/

firefly luciferase ratios.

Quantitative PCR for FOXP3 expression

Total RNA was extracted with Trizol reagent according

to the manufacturer’s guidelines (Invitrogen) and first-strand

cDNA were synthesized by RT (Superscript First-strand Synthesis

System for RT-PCR kit; Invitrogen). Quantitative mRNA

expression was measured by real-time PCR, with PRISM

7900 sequence detection system (PE Applied Biosystems), and

the TaqMan Master mix kit with EF1-a mRNA was used

as an internal control, as well as the GUS gene. The primers

and the internal fluorescence TaqMan probes were designed as

follows:

FOXP3 (forward) 50-TTTCACCTACGCCACGCTCA-30

(reverse) 50-CCAGCTCATCCACGGTCCA-30,

and probe 50-FAM-CCACCTGGAAGAACGCCATCCGC-TAMRA-30;

and EF1-a (forward) 50-CTGAACCATCCAGGCCAAAT-30

(reverse) 50-GCCGTGTGGCAATCCAAT-30,

and probe 50-FAM-AGCGCCGGCTATGCCCCTG-TAMRA-30.

The program used for amplification was: 10 min at 951C

followed by 40 cycles of 15 s at 951C, 1 min at 601C.

Lentiviral vector production

Vesicular stomatitis virus glycoprotein G (VSV-G) pseudo-typed

lentiviral particles were generated by polyethylene imine (Sigma,

St. Louis, MO) co-transfection of 293 T cells with three plasmids,

pMIRNA, pCMVDR8.91 and pMD.G [28].

pCMVDR8.91 is HIV-derived packaging construct that encodes the

HIV-1 Gag and Pol precursors as well as the regulatory proteins Tat

and Rev [29]. Vesicular stomatitis virus glycoprotein G (VSV-G) was

expressed from pMD.G [22]. pMIRNA provided from System Bios-

ciences is a lentiviral-based vector in which an miR precursor mole-

cule has been cloned downstream of the CMV promoter and contains

copepod GFP as a reporter gene; 24h after transient transfection of

293 T cells, viral supernatants were collected, filtered through

0.45mm low protein-binding filters (Nalgene, Rochester, NY), and

concentrated as previously described [30]. The viral pellets were then

resuspended in 1/100 vol of PBS. Viral stocks were stored in aliquots

at �801C and the titers were determined by transducing HeLa T cells

in a limiting dilution assay. Lentiviral vector preparations collected

24h post transfection displayed titers of 108–109 transducing units/

mL in HeLa T cells. No replication-competent virus was detected in

the concentrated lentiviral stocks. A second production cycle was

repeated after 24h, routinely generating lentiviral vector prepara-

tions of approximately 5�107 to 5�108 transducing units/mL.

Lentiviral transduction

Treg and non-Treg purified from cord blood were plated at a

density of 105 cells/well in 12-well tissue culture plates in 1 mL

of RPMI 1640 supplemented with 10% AB serum, 2 mM

L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin

(Lonza Europe, Verviers, Belgium), in the presence of 5mg/mL

PHA-L (Sigma-Aldrich) and 20 U/mL IL-2.

Twenty-four hours after Treg and non-Treg isolation, cells were

exposed to lentiviral vector preparations with MOI 5, in a volume

of 500mL in the presence of 8mg/mL polybrene (Sigma). GFP1

cells were sorted by flow cytometry at day 7 after transduction.

Western blot analysis

A total of 4�106 cells were lysed and subjected to SDS-PAGE using

10% polyacrylamide gels and transferred to polyvinylidene fluoride

membranes (Amersham Biosciences, Uppsala, Sweden) using a

semidry electroblot chamber. Membranes were blocked with TBST

containing 5% BSA overnight at 41C. After blocking, the blots were

incubated with a 1/200 dilution of goat anti-FOXP3 diluted in TBST

for 1h at 251C. Following 1h of incubation with anti-goat peroxidase-

conjugated antibody (Sigma Chemical) at room temperature,

proteins were detected by the electogenerated chemiluminescence



method (Amersham Biosciences), according to the manufacturer’s

instructions.

To confirm sample loading and transfer, membranes were

incubated in stripping buffer and reblocked for 1 h, and then

reprobed using anti-actin (C-2; Santa Cruz Biotechnology).

Detection of mature miR-31 by TaqMan real-time PCR

TaqMan miRNA assays (ABI, Forest City, CA) used the stem loop

method [19, 20] to detect the expression level of mature

miR-31. For RT reactions, 10 ng total RNA was used in each

reaction (15mL) and mixed with the RT primer (3mL). The

RT reaction was carried out at the following conditions:

161C for 30 min; 421C for 30 min; 851C for 5 min, and then held

on 41C. After the RT reaction, the cDNA products were diluted at

5� and 9mL of the diluted cDNA was used for PCR along with

TaqMan microarray assay (1mL) and PCR master mix (10mL). The

PCR was conducted at 951C for 10 min, followed by 40 cycles of

951C for 15 s and 601C for 60 s in ABI’s 7900 real-time PCR system.

The real-time PCR results were analyzed and expressed as relative

miRNA expression of Ct value, which was then converted to fold

changes. RT primer, PCR primers and TaqMan probe for miR-31

were purchased from ABI. RNU44 was used for normalization.

Anti-miRNA transfection

HeLa cells were seeded in p24 plate at 30% confluence with a

serum-free medium without antibiotics the day before transfec-

tion. Fifteen picomole of anti-miR-31 miRNA inhibitor (Ambion)

was transiently transfected in cells using Lipofectamine 2000

according to the manufacturer’s instructions. Cells were incu-

bated in the presence of the specific anti-miR miRNA inhibitor

(anti-miR-31), target miRNA mature sequence: AGGCAAGAUG-

CUGGCAUAGCU and the negative control for 24 h. The Anti-miR

miRNA Inhibitor Negative Control is a random sequence anti-miR

molecule offered for use as a negative experimental control. The

Anti-miR Negative Control has been extensively tested in many

human cell lines and tissues and validated to not produce any

identifiable effect on known miR function.

Statistical analysis

Data are presented as means7SD of five experiments and

analyzed using Student’s t-test. p-Values less than 0.05 were

considered significant.

Acknowledgements: This work was supported by grants from

the Belgian Fonds National de la Recherche Scientifique (FRSM,

Televie), the MEDIC Foundation, les Amis de l’Institut Bordet, the

Van Buuren and Hoguet Foundations. P.R. was supported in part

by a European Union grant ‘‘Cancer immunotherapy’’.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: Ct: cycle threshold � miR: microRNA � HDAC: histone

deacetylase � nTreg: natural Treg � TLDA: TaqMan Low Density Array �UCB: umbilical cord blood � UCBMC: UCB mononuclear cells � 30 UTR:

30 untranslated region

Full correspondence: Dr. Philippe Martiat, Department of Experimental

Hematology, Institut Jules Bordet, Universite Libre de Bruxelles, 121,

Boulevard de Waterloo, 1000, Bruxelles, Belgium

Fax: 132-2-5413453

e-mail: [email protected]

Received: 14/5/2008

Revised: 8/3/2009

Accepted: 9/3/2009



Human natural Treg microRNA signature: Role of microRNA‐31 and microRNA‐21 in FOXP3 expression

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Transcript of Human natural Treg microRNA signature: Role of microRNA‐31 and microRNA‐21 in FOXP3 expression