Enhanced formation and survival of CD4 + CD25 hi Foxp3 + T-cells in chronic lymphocytic leukemia

ORIGINAL ARTICLE: RESEARCH

Enhanced formation and survival of CD4þ CD25hi Foxp3þ T-cells inchronic lymphocytic leukemia

MARGOT JAK1,2, ROGIER MOUS1,2, ESTER B. M. REMMERSWAAL2,

RENE SPIJKER1,2, ANNELIEKE JASPERS1,2, ADRIANA YAGUE1, ERIC ELDERING1,2,

RENE A. W. VAN LIER2, & MARINUS H. J. VAN OERS1

1Department of Hematology, and 2Department of Experimental Immunology, Academic Medical Center, Amsterdam,

The Netherlands

(Received 22 December 2008; revised 2 February 2009; accepted 5 February 2009)

AbstractRecently, it has been described that patients with chronic lymphocytic leukemia (CLL) have increased numbers of regulatoryT (Treg) cells. In the present study, we analysed the mechanism behind Treg cells expansion in CLL. Neither analysis of theT-cell receptor repertoire nor CD45 isoform expression of Treg cells from patients with CLL provided evidence for chronic(tumor) antigenic stimulation as a possible cause for Treg cells expansion in CLL. We found evidence however for increasedformation of Treg cells via CD70 costimulation, because we observed that CD40 ligand activated CLL cells (which might beconsidered a model of lymph node CLL cells) strongly induced CD70-dependent formation of Treg cells. Reversetranscription-multiplex ligation-dependent probe amplification assay expression analysis of 34 apoptosis-regulating genesshowed that in comparison with other CD4þ T-cells, Treg cells from both healthy individuals (HD) and patients with CLLhad a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of Treg cellsin patients with CLL were significantly higher than in HD. Finally, the different apoptotic profile resulted in differences at thefunctional level, because Treg cells from patients with CLL were more resistant to drug-induced apoptosis than Treg cellsfrom HD. In conclusion, Treg cells in CLL may accumulate both by increased formation, facilitated by CD27-CD70interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis because of a shifted Noxa-Bcl-2balance.

Keywords: Chronic lymphocytic leukemia, regulatory T-cells, apoptosis

Introduction

Chronic lymphocytic leukemia (CLL) is charac-

terised by the slow accumulation of mature CD5þ

CD19þ B-cells. Notably, patients with CLL fre-

quently also have increased numbers of circulating

CD4þ and CD8þ T-cells [1,2]. At present, there are

no data supporting CLL-specificity of these ex-

panded T-cell populations. In contrast, we have

shown that patients with CLL can have increased

numbers of cytomegalovirus (CMV)-specific CD8þ

memory effector cells [3].

Recently, it has been described that patients with

CLL have increased numbers of CD4þ CD25bright

regulatory T (Treg) cells, with highest Treg cell

frequencies in progressing patients with extended

disease [4]. Importantly, Treg cells from patients with

CLL show inhibitory function similar to healthy

controls [4]. Treg cells are thought to play an

important role in immune evasion by malignancies

[5,6]. High Treg cell numbers in patients with

malignancies are often associated with poor prog-

nosis [6]. Because high Treg cell numbers in CLL

might negatively affect the course of disease, it will be

important to elucidate the mechanism of this Treg cell

expansion. Most naturally occurring Foxp3þ Treg

cells in adults express markers of primed T-cells,

such as CD45R0. The naıve marker CD45RA, which

Correspondence: Margot Jak, Meibergdreef 9 room K0-154, 1105 AZ, Amsterdam, The Netherlands. Tel: þ31205667732. E-mail: [email protected]

Margot Jak and Rogier Mous contributed equally to this work.

There is an accompanying commentary that discusses this paper. Please refer to the issue Table of Contents.

Leukemia & Lymphoma, May 2009; 50(5): 788–801

ISSN 1042-8194 print/ISSN 1029-2403 online � 2009 Informa Healthcare USA, Inc.

DOI: 10.1080/10428190902803677

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is expressed by a proportion of naturally occurring

Foxp3þ Treg cells, decreases with age [7]. In contrast

to naturally occurring Treg cells which are generated

in the thymus, the majority of Treg cells in healthy

adults are generated in the periphery [8]. These so-

called adaptive or induced Treg cells are likely to be

continuously produced from the memory CD4þ

T-cell pool [9], because most Treg cells possess a

memory phenotype (CD45R0þ) and the T-cell

receptor (TCR) repertoire of Treg cells shows a

high homology to that of the CD4þ memory T-cell

pool. However, the turnover of Treg cells appears to

be much faster than that of memory T-cells [9] and

because in HD there is no steady increase in Treg cell

numbers the increased proliferation rate apparently is

counterbalanced by regulated apoptosis of Treg cells

[10,11]. Thus, the increase of Treg cells in CLL

might be due to either increased formation (either by

increased proliferation or by differentiation of non-

Treg cells into Treg cells), decreased apoptosis or

both. Another possibility of an increase in Treg cells

in the peripheral blood of patients with CLL is

redistribution of Treg cells; by distorting lymph node

architecture tumor cells could drive Treg cells out of

the secondary lymphoid organs into the blood.

In the present study, we have examined potential

mechanisms of increased Treg cell numbers in CLL.

Possible expansion by chronic antigenic stimulation

was evaluated by analysis of the TCR repertoire and

the expression of differentiation markers on Treg

cells. Furthermore, we investigated whether CLL

cells themselves play a stimulatory role in the

formation of Treg cells. Finally, alterations in

apoptosis of Treg cells were studied by expression

profiling of 34 apoptosis-regulating genes as well as

by assessment of Treg cell sensitivity to cytotoxic

drugs.

Methods

Cells from patients with chronic lymphocytic leukemia

and healthy individuals

Peripheral blood was drawn from patients with CLL

(diagnosed according to the NCI-WG guidelines) as

well as from healthy volunteers (blood bank

donors). Peripheral blood mononuclear cells

(PBMC) were isolated and either used immediately

or stored in liquid nitrogen. During all in vitro

experiments, cells were maintained in Iscove’s

modified Dulbecco medium (IMDM: Gibco Life

technology, Paisley) supplemented with 10% heat-

inactivated fetal calf serum, penicillin, gentamycin

and b-mercaptoethanol. All PBMC samples from

patients with CLL contained at least 90% CD5þ/

CD19þ cells as assessed via flow cytometry. The age

range of patients with CLL was 28–86 years

(female: male ratio 1:1.6) and of healthy donors

29–61 years. (F:M ratio 1:0.8). The studies were

approved by the Ethical Review Board of the

Institute and from all participants written informed

consent was obtained. Patient characteristics are

listed in Table I.

Flow cytometry

PBMC were stained using antibodies against CD4,

CD8, CD5, CD19, CD25, CD45RA or CD45R0

and CD127 (all Becton Dickinson, San Jose, CA),

CD70 FITC (clone CLB-2F2) or with antibodies

against CD95 (IQ products, Groningen, the

Netherlands). For intracellular staining, cells were

fixed and permeabilised (eBioscience, San Diego,

CA) and subsequently stained for Bcl-2 (Dako,

Glostrup, Denmark), Ki-67 (BD Pharmingen, San

Jose, CA) and Foxp3 (eBioscience, San Diego, CA).

As isotype-matched control antibody for Bcl-2 and

CD95 we used mouse IgG1 FITC, clone X40 (BD

Pharmingen, San Jose, CA) and for Ki-67 we used

FITC-conjugated mouse IgG1, k isotype control,

clone MOPC-21 (BD Pharmingen, San Jose, CA).

Antibody stained cell samples were analysed by flow

Table I. Patient characteristics.

ID Gender

Age

(years)

Stage

(Rai)

IgVH

mutation

status Prior therapy

1 M 60 2 Mut No Tx

2 M 86 2 Mut No Tx

3 M 52 0 Unmut No Tx

4* F 28 4 Unmut F, CA during analysis


6 F 65 1 Unmut F during analysis


8 M 84 0 Mut No Tx


10* M 35 2 Unmut F,C,R41 year

11 M 62 2 Mut No Tx

12 F 75 0 Mut No Tx

13 M 74 0 Mut No Tx

14 F 59 0 Mut No Tx

15 M 73 1 Mut No Tx

16 F 73 1 Mut CA5 1 year

17 F 59 1 ND No Tx

18 M 78 2 Unmut CA5 1 year

19 F 42 2 Mut CA5 1 year

20 F 62 0 Mut No Tx

21 M 54 2 Mut No Tx

F, female; M, male; Mut, mutated IgVH genes; Unmut,

unmutated IgVH genes; ND, not determined; C, cyclopho-

sphamide; CA, chlorambucil; F, fludarabine; R, rituximab; no

Tx, no therapy.

Patients indicated with an asterisk (*) showed highly progressive

disease.

Enhanced formation of Treg cells in CLL 789

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cytometry with the CellQuest program on a fluores-

cence-activated cell sorting (FACS) Calibur

(Beckton Dickinson).

Isolation of T-cell populations

Thawed PBMC from either HD or patients with

CLL were stained with antibodies against CD4,

CD25 and CD127 (all Becton Dickinson, San Jose,

CA). Subsequently, Treg cells (CD4þ/CD25bright/

CD127low) and non-Treg CD4þ T-cells (CD4þ/

CD257/CD127þ) were obtained by cell sorting

(FACS Aria, Becton Dickinson, San Jose, CA).

The isolated cells were immediately lysed to prepare

RNA or perform protein isolation. Treg cells enriched

populations contained *80% CD4þ/Foxp3þ cells as

assessed by flow cytometry.

Analysis of Vb repertoire

RNA isolated from sorted T-cells was subjected to

template switch-anchored reverse transcriptase-poly-

merase chain reaction by using Super Smart PCR

cDNA Synthesis Kit (BD Biosciences Clontech, Palo

Alto, CA). Vb PCR was performed on amplicons as

described previously [12]. For the spectratyping,

samples were mixed with Genescan-500 ROX size

standards and run on an ABI 3100 capillary

sequencer (Applied Biosystems, Warrington, United

Kingdom) in Genescan mode.

In vitro CD40 ligand stimulation of chronic lymphocytic

leukemia cells

PBMC from patients with CLL (490% CD5þ

CD19þ cells) were stimulated with CD40 ligand

(CD40L) transfected NIH3T3 (3T40L) cells as

described, previously [13]. Briefly, CLL cells were

added to six-well plates coated with g irradiated

(30 Gy) CD40L transfected NIH3T3 cells.

Non-transfected 3T3 cells were used as negative

controls. After 2 days, the CLL cells were gently

removed from the fibroblast layer and used in further

experiments.

Cell stimulation cultures

Cell stimulation cultures (CSC) were performed with

3T3- or 3T40L-stimulated CLL cells antigen pre-

senting cell (APC) and PBMC of a healthy individual

or (autologous) patient with CLL (responders) in a

1:1 ratio (26105 stimulators: 26105 responders).

Cells were cultured in 96-wells plates (Costar,

Corning, NY) in the presence of soluble CD3 mAb

(clone CLB-T3.4/E) [14] and in the presence or

absence of a blocking CD70 mAb (clone CLB-2F2)

[15]. After 4 days, cells were harvested and Foxp3

expression was analysed by flow cytometry as

described above.

Reverse transcription-multiplex ligation-dependent probe

amplification assay

Reverse transcription-multiplex ligation-dependent

probe amplification assay (RT-MLPA) procedure

was performed as described previously [16]. Briefly,

100 ng total RNA as obtained from sorted T-cell

populations was reverse transcribed using a gene-

specific probe mix. The resulting cDNA was

annealed overnight at 608C to the MLPA probes.

Annealed oligonucleotides were covalently linked by

Ligase-65 (MRC, Amsterdam, The Netherlands) at

548C. Ligation products were amplified by polymer-

ase chain reaction (PCR; 33 cycles, 30 s at 958C,

30 s at 608C and 1 min at 728C) using one

unlabelled and one 6-carboxy-fluorescein-labelled

primer (10 pM). PCR products were size separated

on an ABI 3100 capillary sequencer in the presence

of 1 pM ROX 500 size standard (Applied Biosys-

tems, Warrington, United Kingdom). Results were

analysed using the programs Genescan analysis and

Genotyper (Applied Biosystems). Category tables

containing the area for each assigned peak (scored in

arbitrary units) were compiled in Genotyper and

exported for further analysis with Excel spreadsheet

software (Microsoft, Redman, WA). Data were

normalised by setting the sum of all signals at

100% and expressing individual peaks relative to

the 100% value. The obtained expression levels of all

tested genes in Treg cell populations (see isolation of

T-cell populations) were then compared with the

levels found in the non-Treg CD4þ T-cell population

and reflected as relative expression (gene expression

in CD4 set as 1).

Quantative PCR analysis of Noxa expression

Twenty nanograms of RNA extracted from sorted

cell populations (see analysis of Vb repertoire) was

used to synthetise cDNA with superscript II reverse

transcriptase (Invitrogen, Carlsbad, CA). From these

cDNA pools, specific targets were amplified by PCR

performed with Lightcycler FastStart DNA Master

SYBR Green I (Roche Diagnostics, Indianapolis,

IN), using the sense and antisense Noxa primers 50-GGAGATGCCTGGGAAGAAGG-30 and 50-TCA

GGTTCCTGAGCAGAAGAG-30 and the 18S pri-

mers 50-GGACAACAAGCTCCGTGAAGA-30 and

50-CAGAAGTGACGCAGCCCTCTA-30, respec-

tively. The results were normalised to 18 S. Obtained

values for Treg cells were set as 1 and compared with

values of non-Treg cells (relative expression).

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Western blotting

Western blotting was performed as described pre-

viously [17]. Protein samples were separated by 13%

sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis followed by Western blotting. Blots were

probed with the following antibodies or antisera:

polyclonal Mcl-1 (BD Pharmingen, San Jose, CA),

monoclonal anti-Noxa (Imgenex, San Diego, CA),

anti-Bcl-XL (Transduction Laboratories, Lexington,

KY), rabbit-anti-Bcl-2 (Alexis Biochemicals, San

Diego, CA) or antiserum to ß-actin (Santa Cruz

Biotechnology, Santa Cruz, CA).

Drug sensitivity assays

PBMC of both patients with CLL and HD were

incubated with various concentrations of fludarabine

(Sigma-Aldrich, St Louis, MO), Roscovitine (Sigma

Aldrich, St Louis, MO) or agonistic Fas antibody

CH11 (Beckman Coulter, Fullerton, CA). After

24 h, cells were fixed and permeabilised and stained

for CD3, CD4, CD25 (all Beckton Dickinson) and

Foxp3 (eBioscience) to determine the percentage of

Treg cells out of the total CD4 population.

The obtained values were then normalised by

calculating the percentage of Treg cells remaining

after drug stimulation compared with non-stimulated

as follows: (Foxp3þ/(CD4þ)drug stimulated)/(Foxp3þ/

(CD4þ)non-stimulated)6100%.

Alternatively, drug-treated PBMC samples were

fixed and analysed for the presence of fragmented

DNA (permeabilisation buffer containing 0.1 mM

EDTA, 10 mg ml71 propidium iodide and

50 mg ml71 RNase-I) or cleaved caspase-3 (BD

Pharmingen, San Jose, CA) within the Foxp3

positive and negative population.

Statistics

The two-tailed Mann–Whitney U test was used

to analyse differences between two groups.

Alternatively, the Wilcoxon matched paired test was

used to analyse differences between paired samples.

P-values5 0.05 were considered statistically

significant.

Results

Patients with chronic lymphocytic leukemia have

increased numbers of regulatory T-cells in peripheral blood

In agreement with our previous studies in patients

with CLL we found increased numbers of

CMV-specific CD8þ/CD45RAþ/CD277 cells [3]

but no increase in the CMV-associated CD4þ/

CD277/CD287 T-cell population [18] (data not

shown). In addition, we observed an increase in

numbers of CD4þ CD25bright CD127low T-cells

[Figure 1(A)]. This phenotype has been associated

with Foxp3 expression and regulatory function

[19,20]. Indeed, counterstaining with Foxp3 anti-

body showed predominantly Foxp3 positive T-cells

within the CD4þ CD25bright CD127low population

[Figure 1(B)], thereby confirming recent findings

that patients with CLL have increased numbers of

[4] (Treg cells). In line with previous studies [4], we

observed higher Treg cell frequencies (defined as

CD4þ/FoxP3þ cells) in patients with extended

disease (Supplemental Figure S1).

No evidence for predominant antigen involved in

regulatory T-cells formation

A possible mechanism behind Treg cell formation

and/or maintenance is (chronic) antigenic

Figure 1. Analysis of T-cell populations in patients with CLL.

CD4þ T-cells of nine patients with CLL were analysed by flow

cytometry and compared with T-cells of nine age-matched healthy

individuals (HD). (A) Absolute numbers of CD4þ/CD25bright/

IL7Rlow T-cells. (B) Foxp3 counterstaining of CD4þ/CD25bright/

IL7Rlow population of a patient with CLL. After gating on CD4þ

lymphocytes, CD25 was plotted against IL7R (left dot plot). The

CD25bright/IL7Rlow population is indicated with a region mark

(R1). Within R1, the percentage of Foxp3 positive cells was

determined (right dot plot). Dot plots are representative for both

patients with CLL and healthy individuals; the percentage of

CD25bright/IL7Rlow within the CD4þ T-cell population varied

between donors.


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Figure 2. T-cell receptor (TCR) repertoire analysis and phenotype of Treg cells from patients with CLL. A and B: CD4þ/CD25bright/

IL7Rlow (Treg cells) and CD4þ/CD257/IL7Rþ (CD4; non-Treg CD4þ T-cells) from two patients with CLL and two healthy individuals

(HD) were sorted by FACS. RNA isolated from these T-cell populations was used as input for Vb PCR assays. The figures displayed

are representative for both patients with CLL and both healthy individuals, respectively. A: Vb repertoire of each T-cell population

(specified on the left). The bands on gel represent the product of each individual Vb PCR (see bottom of each gel). The markers on the

right indicate the area in which the product for each PCR is expected. *size marker. B: Spectratyping of three randomly chosen Vbfamilies. Each peak represents a CDR3 region with a certain length. On top of the picture, the different Vb families are indicated; the

sorted T-cell populations are indicated on the left. C: PBMC from patients with CLL (n¼ 21) and healthy individuals (n¼ 6) were

stained for CD3, CD4, Foxp3 and CD45R0. The percentage of CD45R0þ cells within the Treg (CD3þ/CD4þ/Foxp3þ) and non-Treg

CD4 (CD3þ/CD4þ/Foxp37) cell population is plotted.

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stimulation. Recently, it has been demonstrated

that upon antigenic stimulation, a limited number

of Treg cell clones arises with the same TCR as

the antigen-specific T-cells from the effector cell

pool [9]. Thus, an antigenic ‘fingerprint’ is present

within both the effector T-cell pool and the Treg

cell population. To see whether a predominant

antigen may be involved in the formation or

maintenance of the Treg cell population in patients

with CLL, we screened the TCR repertoire of

patients with CLL (n¼ 2; patient number 3 and 4,

Table I) as well as the TCR repertoire recovered


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from Treg cells from HD (n¼ 2) and compared

this repertoire to that of non-Treg CD4þ T-cells

(combination of naıve and memory CD4 cells)

from the same individuals. We observed that the

complete range of Vb genes was used in Treg cells

and non-Treg cells from both HD and patients

with CLL [Figure 2(A)]. We randomly chose

three Vb families for fragment length analysis

(spectratyping). Also here, the three randomly

chosen Vb family PCR products showed similar

peak patterns for both non-Treg cells and Treg cells

[Figure 2(B)] in all individuals, making the

involvement of a predominant antigen in Treg

formation in patients with CLL less likely. Only

within the Vb11 family, a discrepant peak was

observed in Treg cells compared with the non-Treg

CD4þ T-cell population, but this peak was present

in Treg cells from both patients with CLL

and HD.

Next, we examined the ‘antigen experience’ of Treg

cells by determining the surface expression of

CD45R0. In line with previous studies [9], we found

that the vast majority of the Treg cells in these adult

individuals have an antigen-experienced phenotype

when compared with non-Treg cells, as characterised

by surface expression of CD45R0 [9] [Figure 2(C)].

We observed no difference in the percentage of

CD45R0 positive Treg cells between HD and patients

with CLL. The percentage of CD45R0þ cells in the

non-Treg cell population was statistically different

between HD and patients with CLL [Figure 2(C)].

This difference could neither be explained by age,

nor by CMV status (data not shown).

CD40 ligand-stimulated chronic lymphocytic leukemia

cells induce Foxp3 expression in CD4þ T-cells in a CD70

dependent manner

Recently, it has been shown that CD70þ non-

Hodgkin lymphoma (NHL) B-cells can induce Treg

cells via CD70 costimulation [21]. In contrast to

peripheral blood CLL cells, CD40L stimulated CLL

cells (which resemble CLL cells from a lymph node

environment [22]) have high CD70 surface expres-

sion [13,23]. Therefore, we hypothesised that in a

lymph node environment CLL cells might facilitate

the formation of Treg cells. To test this, we

performed CSC using CLL cells that were pre-

stimulated with 3T3 or CD40L transduced 3T3 cells

(3T40L; see methods) as APC and PBMC from a

healthy individual or autologous PBMC from

patients with CLL as responder cells. All CSC

were performed in the presence of mitogenic CD3

mAb (see methods). After 4 days, cells were

harvested and analysed by flow cytometry. After

CD40L-stimulation of CLL cells, both the percen-

tage of CD5þ CD19þ CD70þ cells and CD70 mean

fluorescence intensity strongly increased

[Figure 3(A)]. Strikingly, we observed that CD40L-

stimulated CLL cells significantly augmented Foxp3

expression in CD4þ T-cells of a healthy individual

(*P¼ 0.019). Moreover, this augmentation could be

blocked by anti-CD70-antibodies [2F2;

Figure 3(B)]. To test if this effect was also present

in an autologous setting, CD40L-stimulated CLL

cells were used to stimulate autologous T-cells. Also

here, CD40L-stimulated CLL cells augmented

Foxp3 expression in autologous CD4þ T-cells

(n¼ 4), which could again be blocked by anti-

CD70-antibodies [Figure 3(C)]. When paired

CSC’s were analysed, we showed that Foxp3

induction was CD70 dependent; when cultured in

the presence of CD70 blocking antibodies,

Foxp3 induction in CD4þ T-cells of both HD and

patients with CLL was significantly inhibited

[P¼ 0.0156; Figure 3(D)]. As a control we used

anti-CD80-antibodies, which showed no significant

inhibition of Foxp3 induction (data not shown).

High expression of Noxa and low Bcl-2 characterise a

pro-apoptotic profile of regulatory T-cells

Because Treg cells have been described to be highly

susceptible to apoptosis [11], we investigated whether

Figure 3. Cell stimulation cultures (CSC) and Foxp3 induction. CSC were performed with 3T3 or 3T40L stimulated CLL cells (APC) and

PBMC of a healthy individual (HD) or autologous PBMC from patients with CLL (responders) in a 1:1 ratio. Cells were cultured in the

presence of soluble CD3 mAb and in the presence or absence of a blocking anti-CD70 mAb (2F2). Foxp3 expression was analysed after 4

days. A: CD70 expression on CD5þ/CD19þ cells before (t¼ 0) and after 2 days co-culture with 3T3 or 3T40L (n¼3). Left: percentage

CD5þ/CD19þ/CD70þ cells+SEM. Right: CD70 expression of CD5þ/CD19þ/CD70þ cells. Data are presented as mean fluorescence

intensity (MFI)+SEM. Top right: CD70 expression; overlay of 3T3 stimulated CLL cells (light grey line) and 3T40L stimulated CLL cells

(black line). Gated on CD5þ/CD19þ cells. B: CSC with 3T3 stimulated CLL (3T3-CLL) or 3T40L stimulated CLL cells (3T40L-CLL) as

APC and PBMC of HD as responders in presence (grey bars) or absence (white bars) of 2F2 (n¼ 3). Data are presented as percentage

CD4þ/Foxp3þ cells (mean+SEM). (*P¼ 0.019). C: CSC of 3T3 stimulated CLL (3T3-CLL) or 3T40L stimulated CLL cells (3T40L-

CLL) and autologous PBMC of patient with CLL in presence (grey bars) or absence (white bars) of 2F2 (n¼ 4). Data are presented as

percentage CD4þ/Foxp3þ cells (mean+SEM). D: Individual percentages CD4þ/Foxp3þ cells of all performed CSC cultures (HD

responder n¼ 3, CLL responder n¼ 4) with 3T40L-CLL as APC in the presence or absence of the CD70 mAb (2F2). (*P¼ 0.0156

Wilcoxon signed rank test).

3

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this might be related to the expression of pro- or anti-

apoptotic molecules. To establish the ‘apoptotic

profile’ of Treg cells, both non-Treg CD4þ T-cells

and Treg cells from three HD were sorted based on

IL2R and IL7R expression [20]. RNA was extracted

from these T-cell populations and used as input for

RT-MLPA expression analysis to evaluate the

expression levels of 34 apoptosis-regulating genes.


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We found that overall expression profiles in Treg cells

were very similar to those in non-Treg CD4þ T-cells

[n¼ 3; Figure 4(A)]. However, two genes had a

markedly different expression in Treg cells. First, the

levels of the pro-apoptotic BH3-only molecule Noxa

were considerably increased in Treg cells as compared

with non-Treg CD4þ T-cells (2.89 fold increase;

P¼ 0.02). Secondly, in line with previous studies [9]

we found that Bcl-2 expression was significantly

decreased in Treg cells when compared with non-

Treg CD4þ T-cells (3.02 fold decrease; P¼ 0.01).

High mRNA levels of Noxa in Treg cells were

confirmed by quantitative PCR analysis [Figure 4(B)].

Protein analysis subsequently confirmed the elevated

expression levels of Noxa and low expression of

Bcl-2 in Treg cells [Figure 4(C)]. Thus, Treg cells

seem to have a unique apoptotic profile which suggests

enhanced susceptibility to apoptosis induction. In-

deed, spontaneous cell death in purified Treg cell was

higher than in non-Treg CD4þ T-cells [Figure 4(D);

left dot plots, non-treated]. Moreover, when sorted

Treg cells were treated overnight with a moderate

concentration of Roscovitine (a drug that preferably

induces apoptosis in cells with high Noxa levels [24]),

they were much more sensitive to cell death than

non-Treg CD4þ T-cells [Figure 4(D); right dot plots,

Roscovitine]. Moreover, under these conditions,

higher percentages of cleaved caspase 3 and DNA

fragmentation (both classical hallmarks of apoptosis)

were found in Treg cells when compared with non-Treg

CD4þ T-cells [Figure 4(E)].

Regulatory T-cells from patients with chronic lymphocytic

leukemia are relatively protected against apoptosis

Because Treg cells have a highly apoptosis-prone gene

expression profile, the increased number of Treg cells

in CLL might be caused by small alterations in

expression levels of apoptosis-regulating genes.

Figure 5(A) shows the expression levels of 34

apoptosis-regulating genes of both Treg and non-

Treg CD4þ T-cells of two patients with CLL. Similar

to healthy controls Treg cells from patients with CLL

have higher Noxa levels compared with non-Treg

CD4þ T-cells [Figures 5(A) and 5(B)]. Comparing

the expression levels in Figure 4(A) (healthy con-

trols) with levels in Figure 5(A) (patients with CLL),

we further analysed two apoptosis-regulating genes

that showed differences between healthy controls and

patients with CLL (Bcl-2 and IAP1). Comparing

expression levels of these genes in Treg cell popula-

tions to levels in non-Treg cell populations (with non-

Treg CD4þ cells set as 1), we found that Treg cells

from patients with CLL express higher levels of Bcl-2

than Treg cells from HD [Figure 5(B)]. This was

confirmed at the protein level by intracellular staining

[Figure 5(C)]. This supports the notion that Treg

cells from patients with CLL might be relatively

protected against apoptosis, because the elevated

expression of Bcl-2 observed in Treg cells from

patients with CLL might serve to counterbalance

the high expression of Noxa [Figures 4(A), 4(B) and

5(B)]. The mechanism behind high Bcl-2 levels in

Treg cells from patients with CLL remains to be

determined, because in our CSC experiments we did

not observe (CD70 induced) upregulation of Bcl-2 in

Treg or non-Treg CD4þ T-cells (data not shown).

In addition, Treg cells from patients with CLL

seemed to have increased expression of inhibitor of

apoptosis protein 1 (IAP1) [Figure 5(B)], a gene that

has been implicated in apoptotic responses to tumor

necrosis factor [25,26]. On the other hand, we

observed that Treg cells from patients with CLL had

higher expression of Fas/CD95 [Figure 5(D)], a

molecule that has been implied in activation-induced

cell death in T-cells [27]. Finally, Treg cells from

patients with CLL displayed lower cycling activity as

assessed by the percentage of Ki-67 positive cells

[Figure 5(E)].

To test the potential functional consequences of

these changes, we compared Treg cells from patients

with CLL and HD for sensitivity to drug-induced

apoptosis. PBMC from patients with CLL (n¼ 6)

and HD (n¼ 6) were incubated with cytotoxic drugs

or Fas-agonistic antibody and monitored for the

percentage of CD25bright Foxp3þ cells within the

total CD4þ T-cell population [Figure 5(F)]. In Treg

cells from patients with CLL, we observed a strongly

Figure 4. Apoptosis-regulating genes in Treg cells. CD4þ T-cell populations of three healthy individuals (HD) enriched for either Treg

(CD25bright/IL7Rlow) or non-Treg CD4þ T-cells (CD257/IL7Rþ) were isolated by flow cytometry (see methods) and lysed to obtain RNA

and protein content. A: Relative expression levels of apoptosis-regulating genes, measured via RT-MLPA (n¼3; see methods). Results of

individual apoptosis-regulating genes are shown as expression relative to the total signal in the sample. Bars graph represents the

mean+SEM. B: Quantative PCR analysis of Noxa expression in the sorted cell populations. The results are presented as relative expression

compared with the expression levels in Treg cells (black bars); the results were normalised by setting the expression levels obtained for

regulatory T-cells as 1. C: Western blot of pooled protein lysates from Treg and non-Treg CD4þ T-cells. Actin is used as a loading control. D:

Treg and non-Treg CD4þ T-cells (isolated as described above) were incubated in medium (non-treated) or in the presence of CDK inhibitor

Roscovitine (12.5 mm) for 24 h. After 24 h viable cells were identified by flow cytometry (forward-sideward scatter). E: PBMC of a healthy

individual were incubated with Roscovitine (12.5 mm) for 24 h. Subsequently, cells were analysed for apoptosis parameters (caspase-3

cleavage and DNA fragmentation). A Foxp3 antibody was used to identify regulatory T-cells.

3

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decreased apoptosis induction by fludarabine, Ros-

covitine (a drug that acts via the Noxa-Mcl1 axis

[24]) or Fas ligation when compared with Treg cells

from HD. Together, these data support the notion

that reduced apoptosis contributes to the accumula-

tion of Treg cells from patients with CLL.

Discussion

In the present study, we investigated potential

mechanisms behind the expansion of Treg cells in

CLL. We observed that Treg cells from patients with

CLL as well as Treg cells from HD predominantly


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have the phenotype of primed Treg cells (CD4þ

CD25bright Foxp3þ CD45R0þ). Although we did not

find evidence for a predominant (tumor) antigen

driving Treg cell expansion in CLL, our experiments

suggest that T-cell stimulation in a CLL lymph node

environment might result in increased formation of

Treg cells via CD27-CD70 costimulation. Further-

more, we observed that Treg cells (compared with

non-Treg CD4þ T-cells) have a pro-apoptotic phe-

notype characterised by high levels of Noxa and low

expression of Bcl-2. Nevertheless, Treg cells from

patients with CLL seem to be less sensitive to

apoptosis induction than Treg cells from HD,

possibly via increased expression of Bcl-2.

Our finding that Treg cells in patients with CLL are

predominantly CD45R0þ and use a similar TCR

repertoire to non-Treg CD4þ T-cells is in line with a

recent study which indicates that Treg cells arise

continuously from the memory T-cell pool upon

antigenic stimulation [9]. The latter also makes it

tempting to speculate about the involvement of a

predominant tumor antigen/peptide in the formation

of the increased numbers of Treg cells in patients with

CLL. Nevertheless, the TCR repertoire analysis

performed in the current study on Treg cells from

patients with CLL did not support this possibility.

Moreover, also CMV seropositivity did not influence

the percentage of CD45R0þ Treg cells (data not

shown), making a role for this antigen (which has

been demonstrated to influence the CD8þ T-cell

repertoire in CLL [3]) in the formation of Treg cells

in CLL unlikely.

To elucidate the mechanism of increased Treg cell

frequencies in malignancies, an important question

to be answered is whether tumor cells themselves

can promote the expansion and activation of Treg

cells. Recent studies show that NHL B-cells are

powerful inducers of Treg cells [21,28]. Yang et al.

show that CD70þ NHL B-cells induce Treg cell

formation in the lymph node via CD70 ligation. To

test whether CLL cells can also be inducers of Treg

cells, it is important to realise that CLL populations

may be heterogeneous, consisting of quiescent

apoptosis resistant peripheral blood cells, and

actively dividing cells in lymphatic aggregates in

lymph nodes and bone marrow [29]. In vitro, CD40

stimulation of CLL cells increases survival and drug

resistance [13,30]. In vivo, there is histopathologic

evidence that proliferating CLL cells are exposed to

CD40Lþ T-cells in proliferative centers in lymph

nodes [29,31]. After CD40 stimulation, peripheral

blood CLL cells can attract CD4þ CD40Lþ

CCR4þ T-cells by upregulating the T-cell attracting

chemokine CCL22 (CCL22 mRNA is constitutively

expressed in CLL cells purified from involved

lymph nodes) [31]. Furthermore, CD40 stimulation

of peripheral blood CLL cells results in a similar

apoptosis gene expression profile to lymph node

CLL cells [22]. Altogether, these data suggest that

CD40L stimulated CLL cells resemble CLL cells

from a lymph node environment. It has been shown

that in contrast to peripheral blood CLL cells,

CD40L stimulated CLL cells have high CD70

surface expression [13,23]. We show that like

CD70þ NHL cells, CD40L-stimulated CLL cells

are also capable of inducing Treg cells in a CD70

dependent manner. CD40L-stimulated CLL cells

enhanced the formation of Treg cells upon TCR

stimulation [Figures 3(B) and 3(C)], and this effect

could be blocked by CD70 antibodies. In the

absence of TCR stimulation CD40L-stimulated

CLL cells were not able to induce Foxp3 expres-

sion (data not shown). These data suggest that

CD27-CD70 co-stimulation may be an important

step in the formation of Treg cells in B-cell

malignancies. Moreover, if indeed the increased

number of Treg cells in CLL can be explained by

CD70 ligation by CLL cells in vivo, this might also

Figure 5. Analysis of apoptosis sensitivity of Treg cells from patients with CLL. CD4þ T-cell populations of two patients with CLL enriched

for either Treg (CD25bright/IL7Rlow) or non-Treg CD4þ T-cells (CD257/IL7Rþ) were isolated by flow cytometry (see methods) and lysed to

obtain RNA and protein content. A: Relative expression levels of apoptosis-regulating genes, measured via RT-MLPA (n¼ 2; see methods).

Results of individual apoptosis-regulating genes are shown as expression relative to the total signal in the sample. Bars graph represents the

mean; error bars indicate range. B: RNA isolated from Treg and non-Treg CD4þ T-cells (CD4) of two patients with CLL and three healthy

individuals (HD) was used as input for RT-MLPA (see methods). Graphs compose two differently expressed genes (Bcl-2, IAP1) and one

equally expressed gene (Noxa) between Treg cells of healthy controls and patients with CLL. The results are plotted as relative expression,

which is calculated as follows: expression Treg cells population/expression non-Treg CD4þ T-cell population. Gene expression in non-Treg

CD4þ cell is set as 1. Bars represent the mean relative expression; error bars indicate range. C: Bcl-2 staining of PBMC from patients with

CLL (n¼ 21) and healthy individuals (n¼ 11). CD4¼CD3þ/CD4þ/Foxp37; Treg cells¼CD3þ/CD4þ/Foxp3þ. Data are presented as MFI;

samples were standardised by using an isotype-matched control antibody. D: CD95 expression on T-cells from patients with CLL (n¼19)

and healthy individuals (n¼ 5). Data are presented as MFI; samples were standardised using an isotype-matched control antibody. E:

Percentage of Ki-67þ cells in Treg and non-Treg CD4 cells from patients with CLL (n¼ 19) and healthy individuals (n¼10). Cutoff for Ki-67

staining was determined using an isotype-matched control antibody. F: PBMC from patients with CLL (n¼ 6) and healthy individuals

(n¼ 6) were incubated with various concentrations of fludarabine, Roscovitine and Fas-agonistic antibody CH-11 (a-Fas). After 24 h, cells

were harvested and analysed by flow cytometry. Finally, the percentage of Treg cells that remained compared with non-treated samples was

calculated (see methods). Data are presented as mean+SEM.

3

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explain why these Treg cells have increased surface

expression of CD95, because it is known that

CD95 expression is upregulated on CD70 co-

stimulated T-cells [32]. Analysis of number and

phenotype of Treg cells in bone marrow of patients

with CLL showed no difference with peripheral

blood Treg cells (data not shown), indicating that

the bone marrow is probably not the primary site of

Treg cell formation in CLL. Altogether, we there-

fore hypothesise that Treg cell formation in CLL

occurs in lymph nodes where CLL cells may

function as professional APC’s to induce Treg cells,

possibly by CD27-CD70 co-stimulation. The ob-

servation that the highest frequencies of Treg cells

are observed in patients with CLL with extended

disease [4] (and thus with more lymph node

involvement) supports this hypothesis. Moreover,

extended disease may distort lymph node architec-

ture and result in decreased niches for Treg cells,

resulting in Treg cells spill from lymph nodes into

peripheral blood.

In conditions of immune stimulation, Foxp3

might not be a good marker for Treg cells, because

it can be expressed by both regulatory and

activated effector T-cell populations [33]. Foxp3

induction by CD40L-stimulated CLL cells might

therefore be a reflection of activation rather than

induction of functional Treg cells. Distinguishing

Treg cells from activated T-cells in our experiments

however is complicated, given the fact that Treg cell

markers as CD25 and Foxp3 are common activa-

tion markers as well. However, for Treg cell

induction by CD70þ NHL cells it has been shown

that Foxp3 expression remained stable up to 14

days, whereas CD25 expression was transient [21].

Long-term maintenance of Foxp3 expression after

activation would correlate with suppressive capacity

[34]. To further investigate whether CD40L-

stimulated CLL cells are capable of inducing

functional Treg cells that have inhibitory capacity,

new and preferably unique Treg cell surface markers

need to be found.

Next to increased formation in lymph nodes, our

data also suggest that Treg cells in CLL may

accumulate via decreased sensitivity to apoptosis.

The latter may strongly influence the rapid turnover

of Treg cells in vivo, which according to our findings

seems to be facilitated by an altered balance between

two molecules involved in apoptosis regulation:

Noxa and Bcl-2. Therefore, the observed increased

expression of anti-apoptotic Bcl-2 in CLL Treg cells

may counterbalance the high expression of pro-

apoptotic Noxa. In our drug sensitivity assays

[Figure 5(F)] we show that Treg cells from patients

with CLL are less sensitive to fludarabine,

Roscovitine and CD95 ligation (the mechanism via

which Treg cells are thought to be eliminated in vivo

[10]). The increased expression of the potent

anti-apoptotic protein Bcl-2 seems to protect CLL

Treg cells not only against p53-dependent apoptosis

by fludarabine, but also p53-independent apoptosis

by Roscovitine or CD95 ligation. Indeed, over-

expression of Bcl-2 or knockdown of the BH-3 only

pro-apoptotic protein Bid has been shown to protect

against CD95L induced apoptosis in pancreatic islets

cells of mice [35], indicating that in death receptor

induced apoptosis the mitochondrial pathway can be

involved.

Increased frequencies of Treg cells occur in many types

of cancer [36–38]. There is evidence that the presence

of Treg cells in the tumor microenvironment may

affect antitumor responses and promote disease

progression [5,6,39]. This may also be the case in

CLL. Thus, targeting Treg cells in CLL might

influence the course of the disease. Interestingly,

one of the drugs used in the first line treatment of

CLL, fludarabine, has been reported to reduce

frequencies of Treg cells and affect their suppressive

capacity [4]. It has been hypothesised that the effect

of fludarabin on CLL is partially due to its effect on

Treg cell function and frequencies. Our data however

show that Treg cells from patients with CLL are less

sensitive than Treg cells from healthy controls to

fludarabine-induced apoptosis. Moreover, in active

immunotherapy it has been shown that blockade of

CTLA-4 potentates anti-tumor T-cell responses,

possibly by selective targeting of antitumor Treg cells

[40,41]. Our data show that Treg cells from healthy

controls are very sensitive to Roscovitine, a cyclin

dependent kinase inhibitor that targets Mcl-1 and

therefore preferably induces apoptosis in cells with

high levels of its binding partner Noxa [24].

Although Treg cells from patients with CLL are less

sensitive to Roscovitine than Treg cells from healthy

controls, by selectively targeting Treg cells and

inducing apoptosis in CLL cells [42], Roscovitine

could nevertheless be a potent adjuvant drug in active

immunotherapy. Alternatively, in view of the rela-

tively high Bcl-2 expression in CLL Treg cells, it

would also be interesting to monitor Treg cell frequen-

cies and suppressive capacity in patients with CLL

that are being treated with oblimersen, a therapeu-

tical Bcl-2 antisense oligonucleotide [43,44].

In conclusion, Treg cells in patients with CLL appear

to accumulate through increased formation, facilitated

by CD70 ligation by tumor cells in the lymph nodes as

well as by decreased sensitivity to apoptosis due to a

shifted Noxa-Bcl-2 balance. Because the increased

number of Treg might be considered to negatively affect

the course of the disease, targeting either one of the

above-mentioned mechanisms may provide additional

strategies in the treatment of CLL.


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Acknowledgements

The authors are indebted to their patients for their

commitment to this study. They thank the

hematologists of the Department of Hematology of

Meander Medical Centre, Amersfoort, The

Netherlands, for referral of patients. Contribution:

M. J., R. M., E. B. M., R. S., A. J. and A. Y.,

performed experiments, M. J. and R. M. analysed

results and made the figures, M. J., R. M.,

R. A. W. and M. H. J. designed the research,

M. J. and R. M. wrote the article.

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Enhanced formation and survival of CD4 + CD25 hi Foxp3 + T-cells in chronic lymphocytic leukemia

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