cutting edge: il-10-producing regulatory b cells in early human pregnancy

Cutting Edge: IL-10-Producing Regulatory B Cells in Early HumanPregnancyLuise Rolle1, Maryam Memarzadeh Tehran1, Anselm Morell-Garc�ıa1,2, Yanitsa Raeva3, Anne Schumacher1,Roland Hartig4, Serban-Dan Costa3, Federico Jensen1†, Ana Claudia Zenclussen1†

1Experimental Obstetrics and Gynaecology, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany;2University Pompeu Fabra, Barcelona, Spain;3University Women’s Clinic, Otto-von-Guericke-University, Magdeburg, Germany;4Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany

Keywords

Abortion, Breg, hCG, IL-10, pregnancy, TNF-

alpha

Correspondence

Ana Claudia Zenclussen, Experimental

Obstetrics and Gynaecology, Gerhart-

Hauptmann-Str. 35, 39108, Magdeburg,

Germany.

E-mail: [email protected].

†FJ and ACZ share senior authorship.

Submission June 14, 2013;

accepted August 16, 2013.

Citation

Rolle L, Memarzadeh Tehran M, Morell-Garc�ıa

A, Raeva Y, Schumacher A, Hartig R, Costa

S-D, Jensen F, Zenclussen AC. Cutting Edge:

IL-10-producing regulatory B cells in early

human pregnancy. Am J Reprod Immunol

2013; 70: 448–453

doi:10.1111/aji.12157

Problem

The function of IL-10 producing regulatory B cells (Breg) during gesta-

tion is unknown. Here, we aimed to understand their participation in

early pregnancy.

Method

CD19+CD24hiCD27+B cell frequency, measured by flow cytometry,

increased with pregnancy onset but not in the case of spontaneous abor-

tions.

Results

B cells from non-pregnant women cultured with serum from normal

pregnant women produced higher IL-10 levels than those cultured with

serum from spontaneous abortion patients or autologous serum. CD19+-

activated B cells from pregnant women strongly suppressed TNF-a pro-

duction by CD4+T cells when cocultured. We identified hCG as an

important factor regulating the number and function of Breg during

pregnancy.

Conclusions

Breg emerge as important players in pregnancy; they suppress undesired

immune responses from maternal T cells and are therefore important for

tolerance acquisition.

Introduction

Mammalian pregnancy represents a unique process

during a limited period of time, at which the mater-

nal immune system defies a double challenge: to tol-

erate the foreign growing fetus and to be surveillant

against pathogens so to avoid infections that could

affect both mother and fetus.1 Minimal disturbances

to the fine equilibrium between immune activation

and tolerance would compromise fetal survival. High

levels of the pro-inflammatory cytokine, tumor

necrosis factor alpha (TNF-a) jeopardize pregnancy

and interleukin 10 (IL-10) was proposed to be the

most potent anti-inflammatory cytokines counteract-

ing TNF-a.2 B lymphocytes were classically regarded

as effector cells of the adaptive immune system as

they convert into plasma cells and secrete antibodies.

However, new evidences support the existence of a

subpopulation of B cells with immune suppressive

capacity, the so-called regulatory B cells (Breg, 3).

Although different phenotypes for Breg have been

described, they all share the main hallmark of Breg

American Journal of Reproductive Immunology 70 (2013) 448–453

ª 2013 John Wiley & Sons Ltd448

CUTTING EDGE

function: the production of IL-10.3 The main func-

tion of Breg is to maintain the fine immune balance

that is required for tolerance; thus, similar to Treg,

they prevent autoimmunity and help to fight against

infections. Whether they are involved in pregnancy

establishment and/or maintenance is unknown.

Here, we investigated the frequency of Breg dur-

ing early human pregnancy, their functionality as

well as their capacity to control the production of

pro-inflammatory cytokines by activated T cells. We

also characterized one soluble factor that modulates

Breg activity.

Materials and methods

Human Subjects

All experiments including samples from human sub-

jects were reviewed and approved by the Ethics

Committee of the Otto-von-Guericke-University

Medical Faculty (EK28/08 to ACZ). All individuals

were properly informed concerning the purpose of

our research and gave their written consent before

sampling. The characteristics of the recruited partici-

pants are summarized in Table I.

Cell Staining and Flow Cytometry

Leukocytes were isolated from peripheral blood of

normal pregnant women (n = 8), patients suffering

from first trimester abortions (n = 5) and non-preg-

nant women (n = 8). Isolated cells were stained for

CD19 (FITC), CD27 (APC), and CD24 (PercP) or

immunoglobulin isotypes (BD Biosciences) for

30 min at 4°C. About 10,000 events were measured

in all cases. CD19+ cells were gated, and within this

population, the double expression of CD27 and

CD24 was analyzed. Regulatory B cells were defined

as CD19+CD24hiCD27+ as described by Iwata et al.4

For intracellular cytokine detection, cells were first

stained for extracellular markers (CD19 or CD4).

Subsequently, cells were fixed overnight with PFA

1% in PBS and permeabilized with saponin 0.1%.

Fixed cells were stained for IL-10 or TNF-a antibod-

ies (both PE-labeled, BD Biosciences) for 30 min at

4°C. In Fig. 1d, IL-10 expression was measured in

gated CD19+ B cells. In Fig. 2, the percentages of

CD4+TNF-a+ double positive cells were analyzed

within the lymphocyte population.

Cells were measured by flow cytometry (FACSCal-

ibur; BD Biosciences, Heidelberg, Germany). Data

were analyzed with FlowJo (Tree Star).

B-cell Isolation

Total CD19+ B cells were magnetically isolated from

peripheral blood of non-pregnant women by nega-

tive selection using a commercially available mag-

netic separation kit (Miltenyi, Bergisch Gladbach,

Germany). The purity of the CD19+ B cells used in

all the experiments was >95%.

Lymphocytes Isolation and Culture

Total lymphocytes were isolated from peripheral

blood of non-pregnant women. 1 9 106 lympho-

cytes were cultured for 48 hr in the presence of

serum (20%) from either pregnant women or from

patients experiencing abortions. Autologous serum

was used as control. Cells stimulated with CD40L

(5 lg/mL) and CpG (10 lg/mL) served as positive

controls. Cells were harvested, washed, stained for

CD19 and IL-10 and analyzed by flow cytometry.

Cell Sorting

CD19+CD24hiCD27+ IL-10 producing Breg were

sorted from peripheral blood of non-pregnant

women by FACS using a FACS Vantage Diva cell

sorter (BD Bioscience). Data were analyzed

Table I Age (mean � S.D.) and Gestational Age (mean � S.D.) of the Women/Patients Enrolled in this Project

Age Week of pregnancy

First trimester normal pregnant women 25.15 � 4.95 10.13 � 1.54

First trimester spontaneous abortions 28.5 � 2.9 10 � 3.67

Non-pregnant subjectsa 29.34 � 4.7 –

aNone of the donors were under medical treatment neither they suffer pathological condition at the time of the sampling. Pregnant women

presented no proteinuria as well as normal blood pressure at the time of sampling.


ª 2013 John Wiley & Sons Ltd 449

BREG IN PREGNANCY

using FACS Diva software (BD Bioscience) and

FlowJo.

Immunofluorescence

Pure isolated CD19+CD24hiCD27+ IL-10-producing

regulatory B cells were placed on slides and incu-

bated with a mouse anti-human Lh/hCG receptor

antibody (1:100) (Luteinizing Hormone Receptor

(C-2term) ACRIS, Aachen, Germany). Subsequently,

a rabbit anti-mouse FITC secondary antibody (1:100)

was applied. Omitting the first antibody generated

negative controls. Samples were analyzed using a

Zeiss AX 10 microscope (Zeiss, Jena, Germany)

equipped with a HXP-120 Light Source for Fluores-

cence Illumination and Axiovision software.

T- and B-cell Cocultures

CD19+ B cells isolated from non-pregnant or preg-

nant women were cultured for 48 hr in the presence

or absence of CD40L/CpG (1–6 lg/mL), harvested,

washed in fresh medium and further cocultured

(1:1) with CD4+CD25� T cells (isolated from periph-

eral blood of non-pregnant donors by negative selec-

tion using a magnetic separation kit from Miltenyi

Biotec, Germany), in 24-well plates with 500 lL of

RPMI medium supplemented with FBS (10%), peni-

cillin/streptomycin (1%). T cells were activated with

anti-human CD3 (1 lg/mL) antibody, anti-human

CD28 (5 lg/mL) antibody and rhIL-2 (10 ng/mL) for

72 h. Cells were harvested, washed, stained for CD4

and TNF-a and analyzed by flow cytometry.

(a) (b)

(c) (d)

Fig. 1 Representative density dot plots showing the gating of CD19+ cells (a) CD24hiCD27+ (b) regulatory B cells in peripheral blood of non-

pregnant women (non-preg), normal pregnant women at the first trimester (preg, 1st trim) and patients suffering from abortions at the first

trimester of pregnancy (sp abortion). (c) Quantification of CD19+CD24hiCD27+ regulatory B cells. Data are expressed as singly dots with median.

Differences between groups were analyzed by the one-way analysis of variance, followed by a Tukey’s multiple comparison test. P < 0.05 was

considered as statistically significant. (all dot plots with bi-exponential axes are shown, so that they can be better compared). (d) Lymphocytes

were isolated from peripheral blood of non-pregnant women and further cultured with serum from normal pregnant women (first trimester) or

from patients suffering from spontaneous abortions and the expression of IL-10 by CD19+ gated B cells were analyzed by flow cytometry. As

control, autologous serum or CD40L/CpG was used. Data are expressed as mean � S.E.M. and are representative of four experiments performed

in duplicates or triplicates. Differences between groups were analyzed by unpaired t-test. P < 0.05 was considered as statistically significant.


450 ª 2013 John Wiley & Sons Ltd

ROLLE ET AL.

Statistics

Normality was assessed by Kolmogorov–Smirnov

test. If normally distributed, data are shown as

mean � S.E.M. In Fig. 1, data are expressed as scat-

ter dot plots showing medians. Data were analyzed

for statistical significance using Prism 5 software

(GraphPad Software, Inc). Differences between the

means of multiple groups were analyzed by one-way

analysis of variance, followed by a Tukey’s multiple

comparison test. P < 0.05 was considered as statisti-

cally significant and was used as threshold to reject

the null hypothesis.

Results and discussion

Historically, the role of B cells in pregnancy has

been indirectly approached by their capacity to pro-

duce antibodies. Pregnancy-associated protective

antibodies are reportedly increased during the course

of normal pregnancies compared with non-pregnant

women.5,6 Unlike the differences found concerning

the levels of protective antibodies between pregnant

and non-pregnant women, the total numbers of

CD19+ B cells remain unchanged during pregnancy.7

B cells arise now as modulators of the adaptive

immune response, mainly because of their capacity

to secrete cytokines. In particular, a new subpopula-

tion of B cells, the so-called regulatory B cells, has

surged, with multiple effects on health and disease.

Due to their ability to produce IL-10, Breg are sug-

gested to play a critical role in the regulation of the

alloimmune responses, in transplantation tolerance,

in autoimmunity and immunity against infections.3

Their participation in pregnancy remained unex-

plored.

We observed significantly augmented percentages

of CD19+CD24hiCD27+ Breg in normal pregnant

when compared to non-pregnant women (Fig. 1a–c).Notably, women suffering from miscarriages pre-

(a)

(b)

Fig. 2 CD19+ B cells were isolated from peripheral blood of non-pregnant or pregnant women at the first trimester. Isolated cells were stimulated

for 48 hr with or without CD40L/CpG. Afterward, cells were cocultured (1:1) with CD4+CD25� responder T cells isolated from non-pregnant donors

for 48 hr. Cells were harvested, stained for CD4 and TNF-a. (a) shows representative dot plots illustrating the percentages of CD4+TNF-a+ cells. (b)

CD19+ B cells isolated from pregnant women but not from non-pregnant women, with or without further stimulation were able to significantly

inhibit the production of TNF-a by activated CD4+ T cells. Data are expressed as mean � S.E.M. and are representative of three experiments

performed in duplicates or triplicates. Differences between groups were analyzed by the one-way analysis of variance, followed by a Tukey’s

multiple comparison test. P < 0.05 was considered as statistically significant.



BREG IN PREGNANCY

sented significantly lower percentages of

CD19+CD24hiCD27+ Breg than women having nor-

mal pregnancies at first trimester. The levels observed

in these patients were comparable with those mea-

sured in non-pregnant women (Fig. 1a–c). Thus, ourdata indicate that factors present in normal pregnan-

cies but not in failing pregnancies stimulate the

expansion of regulatory B cells.

Beside cellular markers, Breg are defined by their

capacity to produce the potent anti-inflammatory

cytokine IL-10.3 The in vivo role of IL-10 produced

by B cells was first demonstrated in a murine model

of experimental autoimmune encephalomyelitis, in

which the missing IL-10 production by B cells was

proposed to cause the disease.8 Based on that, we

investigated the capacity of serum from normal preg-

nant women or patients suffering from first trimester

abortion to induce the production of IL-10 by B cells

in vitro. We observed that serum from normal preg-

nant women at the first trimester but not from

patients suffering from first trimester abortions sig-

nificantly induced the production of IL-10 by CD19+

B cells when compared to lymphocytes cultured

with autologous serum (Fig. 1d). The production of

IL-10 by B cells upon culture with serum of normal

pregnant women was comparable with the values

observed for the positive control, consisting of B cells

stimulated by CD40L/CpG (Fig. 1). Thus, not only

the number of these cells but also its main mediator,

IL-10 is increased during pregnancy. Thus, it is

tempting to speculate that soluble factors present

during pregnancy boot both Breg number and func-

tion. To explore this possibility, we concentrated in

the most prominent molecule secreted during preg-

nancy: the human chorionic gonadotropin. We ana-

lyzed the expression of the hCG receptor in pure

isolated (sorted) CD19+CD24hiCD27+ regulatory B

cells. We found that nearly all CD19+CD24hiCD27+

cells (~95%) expressed the hCGR (Fig. S1A–B). Most

interestingly, human recombinant hCG was able to

induce in vitro the production of IL-10 by isolated

CD19+ B cells (Fig. S1C). Thus, this hormone is

directly involved in the secretion of IL-10 by B cells

and contributes hereby to the tolerogenic function

of Breg.

Recurrent spontaneous abortions have been

linked to an increase in the levels of the pro-inflam-

matory cytokine TNF-a and blocking TNF-a was sug-

gested as a potential therapy for recurrent

spontaneous abortion.9 IL-10-producing regulatory

B cells were reported as inhibitors of TNF-a secre-

tion by activated T cells in several pathological situa-

tions.10 Here, we analyzed the capacity of B cells

from pregnant and non-pregnant women to control

the production of TNF-a by T cells upon activation.

CD19+ B cells isolated from pregnant women and

activated with CD40L/CpG, known to induce IL-10

secretion (Ywata et al., 2011), significantly inhibited

the production of TNF-a by activated CD4+ T cells

(Fig. 2a,b). Interestingly, B cells isolated from preg-

nant women without CD40L/CpG stimulation

achieved the same effect (Fig. 2a,b). In contrast, nei-

ther CD40L/CpG-activated B cells nor non-activated

B cells from non-pregnant women could inhibit the

production of TNF-a by activated CD4+ T cells

(Fig. 2a,b). Thus, soluble factors present in the sera

from pregnant women boost IL-10 production by

B cells, and this is sufficient to inhibit TNF-a pro-

duction by T cells. We have identified hCG as one

important function in inducing IL-10 production in

B cells. IL-10 producing B cells emerge therefore as

novel players in the acquisition of pregnancy toler-

ance as they can suppress maternal immune cells

with putative detrimental properties. Regulatory

B cells already were shown to efficiently constrain

acute inflammation in several situations. Human

regulatory B cells isolated from healthy donors can

suppress the production of TNF-a by mito-

gen-activated T cells.4 Here, we observed that CD19+

total B cells isolated from pregnant women and acti-

vated ex vivo with CD40L/CpG were able to decrease

the production of TNF-a by mitogen-activated CD4+

T cells in a coculture system. Remarkably, the same

results were obtained with un-stimulated CD19+

B cells isolated from pregnant women confirming

that cells from pregnant women have been naturally

activated, probably by a soluble factor, to produce

IL-10 and exert their regulatory function. In contrast

to the results obtained with sorted CD19+

CD24hiCD27+ regulatory B cells,4 total CD19+ B cells

isolated from non-pregnant women, regardless their

activation status, were not able to inhibit the pro-

duction of TNF-a by T cells. The fact that total B

cells from pregnant women have the same effect as

isolated CD19+CD24hiCD27+ regulatory B cells have

may be explained by the elevated proportion of reg-

ulatory B cells in these samples. This supports the

concept of regulatory B cells contributing to the

maintenance of immune tolerance during preg-

nancy. The inhibition of TNF-a production by

maternal T cells represents an important mechanism

of immunoregulation.2 It is known that this potent


452 ª 2013 John Wiley & Sons Ltd

ROLLE ET AL.

inflammatory cytokine is augmented in recurrent

spontaneous abortions.2

In conclusion, we introduce a new concept about

how immune balance during pregnancy is achieved.

Based on our data, pregnancy establishment and

particularly the raise in hCG levels induce an expan-

sion of regulatory B cells and boost their IL-10 pro-

duction which contributes to fetal tolerance, for

example, by controlling the production of TNF-a by

T cells. This novel concept is worth to be investi-

gated in experimental models with the final aim to

create strategies for restoring the immune balance in

those patients with spontaneous abortions owed to

an incomplete immune tolerance.

Acknowledgments

We are very grateful to Markus Scharm for his invalu-

able assistance in all experiments. We especially thank

all of the participants of this project who kindly

donated blood for this study. We also especially thank

the medical and nonmedical staff from the Women’s

Clinic of Magdeburg for their invaluable support

collecting the samples involved in this work.

Funding

This study was financed by grants from the Deutsche

Forschungsgemeinschaft to A.C.Z. (ZE 526/7-1). This

work is part of the MD thesis of Luise Rolle. FJ is

funded by the Fritz Thyssen Foundation, and AMG

was supported by the University Pompeu Fabra. None

of the authors has any conflict of interest to declare.

Disclosure Statement

None of the authors have financial disclosures to

report.

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Supporting Information

Additional Supporting Information may be found in

the online version of this article:

Figure S1. (A) representative pseudo-color dot

plots depicting the expression of hCGR on pure cell-

sorting isolated CD19+CD24hiCD27+ regulatory

B cells from non-pregnant donors. (B) Representa-

tive pictures showing the expression of hCGR on

CD19+CD24hiCD27+ regulatory B cells by immuno-

fluorescence (left picture) or negative control (right

picture). (C) Total lymphocytes were isolated from

non-pregnant donors and further cultured with

human recombinant hCG with or without addition

of CD40L/CpG. Percentages of CD19+IL10+ cells

within lymphocyte population was analyzed by flow

cytometry. Data are expressed as mean � S.E.M.

and are representative of at least four experiments

performed by duplicates. Differences between groups

were analyzed by the one-way analysis of variance,

followed by a Tukey’s multiple comparison test.

P < 0.05 was considered as statistically significant.



BREG IN PREGNANCY

cutting edge: il-10-producing regulatory b cells in early human pregnancy

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