effect of parity on memory and regulatory t cells subsets...
TRANSCRIPT
Effect of parity on memory and regulatory
T cells subsets in the decidua Scientific report
Author:
L.P.Zijlker
S2485907
Supervisors:
Dr. M.M. Faas1
Dr. J.R. Prins2
Department: 1Department of Obstetrics and Gynecology
2Department of Pathology and Medical Biology
University Medical Center Groningen
University of Groningen
Research period:
21-09-2016 – 24-05-2017
2
3
Abstract
Introduction. During pregnancy, the maternal immune system acquires a unique state of
tolerance for the allogeneic fetus. The exact mechanisms responsible for this tolerance remain
unclear, however, modulation of the T-lymphocyte response at the fetal-maternal interface
appears to be important for a successful pregnancy. From previous research it can be
concluded that the maternal immune system can specifically recognise fetal antigens and
forms memory cells possibly specific for these fetal antigens. Furthermore, the risk of
developing immunological pregnancy complications is lower in subsequent pregnancies. In
this research we aimed to determine the influence of parity on memory and regulatory T cell
subsets in the human decidua.
Methods and Materials. Lymphocytes were enzymatically isolated from the decidua of
nulliparous (n=4), primiparous(n=5) and multiparous women (n=6). The lymphocytes were
stained with an antibody mix for characterisation of memory T cells and Tregs. Analysis was
done using flow cytometry.
Results. We found highly activated memory T cell subsets, but no significant differences
between the three groups . We found significantly higher proportions CD4+ and CD8+
activated Tregs in primiparous and multiparous women as well as suppressive CD4+ and
CD8+ Tregs. We also found significantly higher proportions of CD4+ and CD8+ memory
Tregs in primi- and multiparous women.
Conclusion. We conclude that parous women show a more activated and suppressive
regulatory T cell population at the fetal-maternal interface, as well as higher proportions
memory regulatory T cells. Further research is needed to investigate the effect of these
changes in regulatory T cell subsets on the development of immunological pregnancy
complications.
4
Contents
Abstract ..................................................................................................................................... 3
Contents ..................................................................................................................................... 4
List of abbreviations ................................................................................................................. 6
1. Introduction .......................................................................................................................... 7
1.1 Immune associated complications of pregnancy .............................................................. 7
1.2 Immunology of pregnancy ................................................................................................ 7
1.2. Fetal-maternal interface ................................................................................................... 8
1.3 Adaptive immune system ................................................................................................. 8
1.3.1 Regulatory T-cells ...................................................................................................... 9
1.3.2 Memory T-cells .......................................................................................................... 9
1.4 Aim of this study. ........................................................................................................... 10
2. Methods and Materials ...................................................................................................... 11
2.1 Population ....................................................................................................................... 11
2.2 Anatomy of the placenta ................................................................................................. 11
2.3 Samples ........................................................................................................................... 11
2.4 Analysis .......................................................................................................................... 14
2.5 Gating strategy ................................................................................................................ 14
2.6 Statistics .......................................................................................................................... 16
3. Results ................................................................................................................................. 17
3.1 Demographics and clinical characteristics ..................................................................... 17
3.2 Memory T cells ............................................................................................................... 18
3.2.1 CD4+ memory cells ................................................................................................. 18
3.2.2 Activated CD4+ Memory T Cells ............................................................................ 19
3.2.3 CD8+ Memory T cells ............................................................................................. 20
3.2.4 Activated CD8+ memory T cells ............................................................................. 21
3.3 Tregs ............................................................................................................................... 22
3.3.1 CD4+ Treg cells ....................................................................................................... 22
3.3.2 CD4+ Memory Tregs ............................................................................................... 23
3.3.3 CD8+ Treg cells ....................................................................................................... 24
3.3.4 CD8+ memory Tregs ............................................................................................... 25
4. Discussion ............................................................................................................................ 26
4.1 Memory T cells ............................................................................................................... 26
5
4.2 Tregs ............................................................................................................................... 27
4.2.1 Memory Tregs .......................................................................................................... 27
4.2.2 Suppressive Tregs .................................................................................................... 28
4.2.3 Activated Tregs ........................................................................................................ 28
4.3 Strengths and limitations ................................................................................................ 29
4.4 Conclusion ...................................................................................................................... 30
4.5 Future perspectives ......................................................................................................... 30
References ............................................................................................................................... 31
Acknowledgements ................................................................................................................. 35
Abstract Nederlands .............................................................................................................. 36
Appendix ................................................................................................................................. 37
Protocol Enzymatic isolation of lymphocytes from decidua tissue ...................................... 37
6
List of abbreviations
APC Antigen presenting cell
B cell B-lymphocyte
BMI Body mass index
CM cell Central memory cell
CTLA-4 Cytotoxic T-lymphocyte-associated protein 4
DC Dendritic cell
EM cell Effector memory cell
FMO Fluorescence minus one
FOXP3 Forkhead box P3
FSC Forward scatter
HLA Human leukocyte antigen
IDO Indoleamine 2,3-dioxygenase
IQR Interquartile range
IUGR Intrauterine growth restriction
MHC Major histocompatibillity complex
MP Multiparous
NP Nulliparous
PBS Phosphate buffered saline
RM cell Tissue-resident memory cell
PP Primiparous
rpm Rounds per minute
RPMI 1640 Roswell Park Memorial Institute 1640 medium
SD Standard deviation
SSC Side scatter
RT Room temperature
T cell T-lymphocyte
TCR T cell receptor
Th T helper cell
Treg T regulatory cell
TRM Tissue-resident memory T cell
7
1. Introduction
Pregnancy is a unique phenomenon, which presents an immunological challenge for the
maternal immune system. Under normal circumstances, the immune system rejects allogeneic
cells, such as transplants, tumours and microbes to protect the body from potential harm(1). A
fetus expresses HLA-antigens which are both maternal and paternal derived and the fetus is
therefore considered to be semi-allogeneic for the maternal immune system(2). However, the
maternal immune system does not reject the fetus but instead adapts and develops tolerance
towards it. Even while the maternal immune system tolerates the semi-allogeneic fetus, it can
still fight pathogens and infections, which illustrates the careful regulation of anti- and pro-
inflammatory responses by the immune system during pregnancy(2).
Though numerous studies have been conducted to identify the mechanisms responsible for the
development of tolerance of the maternal immune system towards the fetus, the exact
pathways are still unknown.
1.1 Immune associated complications of pregnancy
An inadequate adaptation of the maternal immune system during pregnancy is associated with
pregnancy complications such as preeclampsia, intrauterine growth restriction (IUGR) and
recurrent miscarriage(3–6). Preeclampsia is one of the leading causes of perinatal, maternal
and fetal morbidity and mortality, complicating 2-8% of pregnancies and contributing 16% of
maternal deaths in developed countries(4). The incidence of preeclampsia is higher in women
who are pregnant for the first time (primiparous women) compared to multiparous women
who have already experienced a normal pregnancy. However multiparous women have the
same risk of developing preeclampsia as a primiparous women when one of their subsequent
pregnancies is from a different father(3,7). Therefore it could be hypothesized that not the
state of pregnancy is memorized, but specifically paternal antigens.
This hypothesis is also supported by findings that prolonged exposure to seminal fluid, and
thus to paternal antigens, prior to the pregnancy is associated with a reduced risk at
preeclampsia and IUGR(8,9). How the memorizing competence of the maternal immune
system contributes to a healthy pregnancy has not been fully elucidated.
1.2 Immunology of pregnancy
The first hypothesis that tried to explain the unique phenomenon of tolerance for the
allogeneic fetus by Medawar (1953) was threefold: an absolute physical barrier between the
mother and the fetus, immunologically immature fetal antigens and inertness of the maternal
immune system prevent an attack on the fetus by the maternal immune system(10).
However this threefold hypothesis has been proven invalid over time. Although the fetal-
maternal barrier minimises immunological contact, it is not absolute. Fetal trophoblast cells
invade the uterine wall of the mother which then forms the maternal part of the placenta, the
decidua(11). Maternal immune cells which reside in the decidua therefore have direct access
to fetal antigens(12). Immunological contact between the mother and the fetus is also
demonstrated by research that found fetal antibodies to circulate in the blood of the mother,
even for years after pregnancy (microchimerism)(13). Maternal lymphocytes are able to
recognize fetal antigens through indirect presentation by antigen-presenting cells (APC)(14).
Therefore immaturity of fetal antigens is an insufficient explanation for the tolerance of the
8
fetus. As for the inertness of the maternal immune system, studies have shown that activated
T cells specific for fetal antigens circulate in the peripheral blood of the mother(15–17)
1.2. Fetal-maternal interface
As concluded above, the maternal immune system has access to fetal antigens and can
actively recognize it. This suggests there are additional mechanisms which help to evade an
immune response of the maternal immune system against the fetus.
The fetus is connected to the mother by the placenta. In the placenta, nutrients, oxygen and
waste products are exchanged between the blood of the mother and the fetus(11). As
mentioned before, during gestation fetal trophoblast cells invade the uterine wall of the
mother. The multiple sites where fetal trophoblast cells are in direct contact with maternal
tissue are referred to as the fetal-maternal interface(12). At the fetal-maternal interface, the
maternal immune system is exposed to fetal antigens. The placenta plays an important role in
preventing rejection of the allogeneic fetus. Amongst others, cells of the innate immune
system are important in preventing an immune attack by the mother(12). The innate immune
system comprises the first, non-specific but quick response against microbes. Innate immune
cells such as macrophages, natural-killer cells (NK cells) and dendritic cells are abundant in
the placenta and essential for a successful pregnancy(18).
1.3 Adaptive immune system
As opposed to the innate immune system, the adaptive immune system is more specific but
has a slower response against foreign antigens. The adaptive immune system can be divided
into the humoral and the cellular response(1). The humoral response is mediated by B-
lymphocytes and focuses on extracellular immunity. The main mechanism of the humoral
response is the production of antibodies that can specifically attack circulating foreign
antigens, upon recognition by B-cells(1). The cellular immune response is mediated by T-
lymphocytes and comprises the intra-cellular immunity. T cells are able to recognize infected
cells and mediate in the destruction or actively destroy the infected cells themselves(1).
The T cell receptor (TCR) of T-lymphocytes recognizes antigens when presented on a major
histocompatibility complex (MHC). Within the T-lymphocyte population two subsets can be
distinguished, the CD4+ T-cells that are able to recognize antigens presented on a MHC-II
complex and CD8+ T cells that recognize antigens presented on a MHC-I complex. CD4+ T
cells, or T-helper cells release cytokines that regulate the immune response. CD8+ T cells, or
cytotoxic T cells are able to kill infected or damaged cells(1).
CD4+ T cells can be divided in different subsets, amongst others T-helper 1 (Th1), T-helper 2
(Th2) and T-helper 17 cells (Th17). Th1 cells stimulate a pro-inflammatory immune response
and are involved in auto-immune reactions while Th2 cells have an anti-inflammatory
function and are involved in allergic reactions. Th17 cells, like Th1 cells, mediate an
inflammatory response and are involved in auto-immunity. A shift in the balance between
Th1 and Th2 towards a Th2 dominance has been demonstrated in normal pregnancy(19). In
women with preeclampsia this Th1-Th2 shift was found absent, or even a shift towards Th1
was found(20). It was hypothesized that the Th1 down regulating and anti-inflammatory Th2
dominance was responsible for developing tolerance for the allogeneic fetus(21). However,
recent research found that this explanation does not fully cover the development of maternal
tolerance during the pregnancy. This was demonstrated amongst others by Th2 cytokine (Il-4
and Il-10) deficient mice which do not show a disturbed pregnancy(21–23).
9
1.3.1 Regulatory T-cells
Regulatory T cells (Tregs) are specialised T cells that are potent immune-response
modulators. They are able to supress a cytotoxic T cells response, and are identified by the
expression of the transcription factor Foxp3(24). Tregs are involved in immunological self-
tolerance but there is also accumulating evidence that they play a key role in the
immunological acceptance of the allogeneic fetus(25–28). Tregs are able to suppress the Th1
and Th17 response, which has been noticed to be increased in women who suffered recurrent
miscarriage, and low percentages of Tregs were found in the blood of preeclamptic
women(27,29).
In healthy pregnancies, research has found high concentrations of CD4+ Tregs at the fetal-
maternal interface, that were able to suppress fetus-specific and fetus-nonspecific immune
responses(30). Furthermore, elevated levels of fetal-specific CD4+ Tregs that persist long
after pregnancy and a rapid expansion of Tregs during a subsequent pregnancy, indicate the
development of Treg memory cells(31).
1.3.2 Memory T-cells
Upon activation by an antigen, a small proportion of T cells differentiate into a memory cells.
These memory cells recognize the antigen at a second encounter and stimulate an even more
effective immune response. Different subtypes of memory T cells can be distinguished: the
effector-memory cells (EM), central-memory cells (CM) and tissue-resident memory cell
(TRM)(32)(Fig1). EM cells are memory cells that migrate to the site of inflammation and
show immediate effector function. CM cells reside in secondary lymph nodes and are not only
able to rapidly proliferate upon antigen stimulation, but also to renew themselves(33). Tissue-
resident memory cells are a special population of memory cells that remain permanently in
peripheral tissue even after infection(32).
Memory T cells express the cell surface marker CD45RO(34). Within the memory T cell
population CM cells can be identified by the chemokine receptor CCR7(32). When activated
they lose CCR7 and proliferate to EM cells, which express CD69(32). The TRM cells are
identified using the cell surface markers CD69 and CD103(Fig. 1)(32)(35).
Figure 1. Proliferation patterns of memory T cells(35)
10
CD8+ EM cells are the most abundant type of T cells in the decidua(36,37). The
accumulation and activation of CD8+ EM cells found in the decidua during pregnancy, is
however not seen in peripheral blood(38). A previous study showed that in peripheral blood,
levels of CD4+ EM cells were higher during pregnancy compared to non-pregnant women.
The CD4+ EM, CM and activated memory T cells were elevated in parous women compared
to women who have never been pregnant (nulligravid women)(38). These findings support the
hypothesis that pregnancy is an immunological challenge which triggers the generation of
memory cells. The alteration of the memory T cell population could be an explanation for the
higher incidence of preeclampsia in primiparous women compared to multiparous women.
How one, or multiple pregnancies influence the memory T cell population at the fetal-
maternal interface, however is not fully known.
1.4 Aim of this study.
In this study, we aim to examine the effect of parity on the different subsets and
characteristics of memory T cells in human decidual tissue. The populations of memory T
cells in nulliparous, primiparous and multiparous women will be compared. Additionally, the
regulatory T cell populations will be determined and compared.
In previous research, elevated levels of CD4 central- and effector-memory cells have been
reported in peripheral blood after pregnancy(38). We therefore hypothesized that memory T
cell subsets are generated during first pregnancy and can rapidly expand during subsequent
pregnancy. Following these data we hypothesised to find higher levels of memory T cells with
tolerance competence in primiparous and multiparous women compared to nulliparous
women.
More knowledge about the memory T and Treg population at the fetal-maternal interface will
help to elucidate the mechanisms responsible for maternal immunological tolerance for the
allogeneic fetus. This knowledge could be implemented in the development of treatment or
prevention of immune associated adverse pregnancy outcome, preeclampsia, IUGR and
recurrent miscarriages.
11
2. Methods and Materials
2.1 Population
The placentas of healthy women aged between 18 and 40, who were scheduled for an elective
caesarean section at >37 and < 42 weeks of gestation were included in this study. Women
with an immune deficiency, or those who used medication during their pregnancy were
excluded. All pregnancies were conceived in a natural way and if multiparous, all the
pregnancies were conceived by the same father according to the mothers. Four nulliparous,
five primiparous and six multiparous women were included. The women were classified
according to their parity at the time of inclusion. The included women that were expecting
their first child were classified as nulliparous, women expecting their second child were
classified as primiparous and all women pregnant for the third time or more, were classified
as multiparous. All women gave
informed consent and the study was
approved by the Medical Ethics
Committee of the UMC Groningen.
2.2 Anatomy of the placenta
The placenta consists of a fetal and a
maternal side. The maternal side,
called the decidua, can be divided in
the decidua parietalis and the decidua
basalis. The decidua parietalis is
maternal part which is connected to
chorioamniotic membrane and the
decidua basalis is the most outer part
of the basal plate which is connected
to fetal trophoblast cells (Fig. 2).
Isolation of lymphocytes from the
decidua parietalis and basalis enables
analysis of maternal lymphocytes that
were exposed to fetal antigens.
2.3 Samples
The placenta was collected
immediately after the caesarean-
section and brought to the lab.
Samples were taken from the maternal parts of the placenta.
The decidua basalis was obtained by cutting of the cotyledons of the basalis plate, which then
were washed in phosphate buffered saline (PBS) (PBS, without Mg and Ca, Lonza), and
cutting of the villi to get rid of the trophoblastic tissue. In order to obtain the decidua
parietalis, the amnion was removed and the chorioamniotic membrane was cut off the basal
plate and washed in PBS to rinse off blood clots. Small blood vessels were removed using
forceps. The decidua parietalis was scrapped of the chorion using a Costar® cellscraper
(Corning Incorporated).
Both tissues were minced and washed again. The tissue was then transferred into C-tubes
(Miltenyi Biotec) at 5ml tissue per tube and 10ml of StemProAccutase (Life Technologies)
was added.
Figure 2. Anatomy of the placenta
12
The decidual tissue was mechanically dissected using
a Gentlemacs dissociator (Miltenyi Biotec) and
incubated in accutase in a water bath at 37°C for 45
minutes for enzymatic dissociation of the decidua
tissue. The enzymatically dissolved tissue was then
filtered through a 70 µm strainer with PBS. The
resulting cell suspensions were washed and 10 ml of
RPMI was added. For separation of the lymphocytes
from the other cells, a percoll gradient was used. The
gradient consisted of three layers at different
dilutions: 10 ml 40% (ρ=1.050 g/ml), 12,5 ml 45%
(ρ=1.056 g/ml) and 10 ml 68% (ρ=1.084 g/ml). To the
10 ml 40% dilution, 10 ml suspension was added and
layered on top of the 68% and 45% percoll dilution.
The percoll gradient was centrifuged (Thermo Fisher
Scientific, heraeus multifuge X3R) at 2000 rpm for 30
minutes without brake.
The lymphocyte layer between the 40% and 45%
dilution layers was taken off (Fig. 3) and the resulting
cell suspensions were washed with PBS. The number
of cells were counted using a coulter counter (Beckman).
Staining
The cells were transferred to a well plate at a million cells per well. First the cells were
blocked with 50 µl Fc-Block (BD Bioscience) and mouse serum (Sanquin, The Netherlands)
in PBS for 10 minutes at room temperature, to reduce aspecific binding of antibodies.
Subsequently the cells were stained with 50 µl (0.43 µg) FVS 620 (BD Biosciences) at room
temperature to distinguish between live and dead cells.
To identify Treg and memory T cell subsets, two different antibody mixes were used, both
containing 8 antibodies. 50 µl of each extracellular antibody mix (BD Biosciences) was added
to the cells and incubated for 30 minutes at 4°C (Table 1).
Figure 3. Percoll layers
13
Table 1. Antibody panels (BD Biosciences)
A. Memory T cell antibody panel.
Antibody Concen
tration
Clone Fluorochro
me
Intra-/
extra-
cellular
Marking
CD4 1:100 SK3 APC-H7 Extra-
cellular
T-helper cells
CD8 1:200 RPA-T8 PE-Cy7 Extra-
cellular
Cytotoxic T-cells
CD45RO 1:20 UCHL-1 BV510 Extra-
cellular
Memory T-cells
CD103 1:50 BER-ACT8 BV421 Extra-
cellular
Tissue resident T-
memory cells
CCR7 1:10 150503 PE Extra-
cellular
Central memory T-
cells
CD69 1:20 FN50 APC Extra-
cellular
Activation maker
Foxp3 1:20 236A/E7 AF488 Intra-
cellular
Treg cells
FVS 620 1:50 PerCP-
Cy™5.5
Extra-
cellular
Viability
B. Regulatory T cell antibody panel.
Antibody Concen
tration
Clone Fluorochrome Intra-/
extra-
cellular
Marking
CD4 1:100 SK3 APC-H7 Extra-
cellular
T-helper cells
CD8 1:200 RPA-T8 PE-Cy7 Extra-
cellular
Cytotoxic T-cells
CD45RO 1:20 UCHL-1 BV510 Extra-
cellular
Memory T-cells
CD127 1:20 hIL-7R-
M21
PE Extra-
cellular
Negative for Treg
cells
HLA-DR 1:200 646.6 APC Extra-
cellular
Treg memory cell
CTLA-4 1:20 BNI3 BV421 Intra-
cellular
Suppressive function
marker
Foxp3 1:20 236A/E7 AF488 Intra-
cellular
Treg cells
FVS 620 1:50 PerCP-
Cy™5.5
Extra-
cellular
Viability
14
Following extracellular staining, the cells were washed with PBS and fixed and permeabilised
with BD fix/perm for 40 minutes on ice. After permeabilisation the cells were washed twice
with BD Perm/wash buffer. The lymphocytes were then stained with an intracellular
antibody-mix for 30 minutes on ice before they were again washed twice with BD Perm/wash
buffer. The samples were diluted in 150 µl PBS before analysis (see appendices for full
protocol).
2.4 Analysis
The samples were analysed immediately after staining using a FACSVerseTM Flow
cytometer and BD FACS SuiteTM software (BD Biosciences). Compensation settings were
set using beads stained with a single antibody (UltraComp eBeads, eBioscience).
2.5 Gating strategy
To identify different sub-populations of T-cells the data were analysed using FlowJo v10
software. Gates were set using an unstained control, due to the low amount of isolated cells
gates could not be set using isotype controls or fluorescence minus one (FMO). However,
previous research in our group has shown that isotype controls did not vary much between the
experiments(38). First, a gate was set on the lymphocytes in a FSC/SSC scatterplot based on
size and granularity (Fig. 4A, Fig. 5B). In the lymphocyte population, the live lymphocytes
were selected in a histogram by setting a gate on the FVS negative cells (Fig. 4B, Fig. 5B).
The CD4+ and CD8+ T-cells were then displayed and gated in a scatterplot (Fig. 4C, Fig.
5C).
Memory T-cells
Within the CD4+ and CD8+ populations, the Foxp3- cells (Fig. 4D) and CD45RO+ cells (Fig.
4E) were selected in histograms to identify the memory T-cells. To identify different subsets
of T-memory cells, CD69 was used as an activated T-cell marker, CD103 as a tissue-resident
T-memory cell marker (Fig. 4F) and CCR7 was used as a central memory T-cell marker (Fig.
4G). This memory panel also permits identification of Tregs through selection of the Foxp3+
cells.
Regulatory T-cells
The regulatory T-cells can be identified by selecting the Foxp3+ and CD127- cells in the
CD4+ and CD8+ populations (Fig. 5D). CD45RO was used as a Treg memory cell marker,
CTLA-4 for suppressive antigen-experienced Tregs and HLA-DR as an activation marker
(Fig. 5E, Fig. 5F).
15
a b c
d e f
g h i
Figure 4. Gating strategy to identify memory T-cells. FSC/SSC scatterplot of all events with gate set around
lymphocytes (A). The FVS negative cells are selected in a histogram to select the vital cells (B) In a scatterplot CD4+
and CD8+ are distinguished (C). The Foxp3 negative population is selected in a histogram (D). The CD45RO
population is selected in a histogram (E). In a scatterplot the CD69, CD103 and CCR7 populations can be distinguished
(F,G,H). The CCR7 and CD69 positive populations are shown in a scatterplot.
16
2.6 Statistics
Data analysis was performed using IBM SPSS Version 23 data editor for Windows and
GraphPad Prism 5.04 software. Because of the small sample size, non-parametric distribution
of all data was assumed. Statistical differences between the three groups were tested using the
Kruskal-Wallis test where after the groups were compared pairwise using the Mann-Whitney
U test. Additionally the PP and MP group combined was compared to the NP group using a
Mann-Whitney U test. Differences between groups were considered statistically significant if
p<0.05. Because of the small groups, means instead of medians with interquartile range are
shown in the graphs.
Figure 5. Gating strategy to identify Tregs. FSC/SSC scatterplot of all events with gate set around lymphocytes (A).
The FVS negative cells are selected in a histogram to select the vital cells (B) In a scatterplot CD4+ and CD8+ are
distinguished (C). The Foxp3 positive population and CD127 negative population is selected in a histogram (D). In a
scatterplot the HLA-DR and CTLA-4 positive populations can be distinguished (E) and the CD45RO positive group is
selected in a scatterplot (F). In the CD45RO positive population, the HLA-DR and CTLA-4 population are shown (G)
a b c
d e f
g
17
3. Results
3.1 Demographics and clinical characteristics
To examine the effects of parity on T cell subsets, 4 nulliparous, 5 primiparous and 6
multiparous women were included in this study. One decidua basalis sample in the
multiparous group was excluded because of incorrect handling in the lab. The patient
characteristics of these groups are shown in table 2.
There were no significant differences in maternal age or gestational age between the three
groups. The BMI of women in the nulliparous (NP) group was significantly lower than those
in the primiparous (PP) and multiparous (MP) group (p<0.05), however, there was no
significant difference between the PP and MP group. The birth weight of the babies in the NP
group was significantly lower compared to those in the PP group (p=0.05). No other
significant differences were found.
In all three groups there was one person who reported smoking during her pregnancy, none of
the women reported using alcohol during the pregnancy. All women received the same
antibiotics prior to the caesarean section. The pregnancies of multiparous women were
conceived by the same father as the previous pregnancies.
Table 2. Characteristics of the patient groups.
Nulliparous
(N=4)
Primiparous
(N=5)
Multiparous
(N=6)
Maternal age (years) 28 (22.8 ± 31.8) 30 (27 ± 34.5) 35 (27 ± 39)
BMI (kg/m2) 23.88 (20.6 ± 26.2)* 28.63 (27.1 ± 34.9) 32.98 (27.7 ±36.3)
Gestational age
(days)
275 (266 ± 277.3) 275 (270 ± 280) 274 (273 ± 275)
Parity 0 1 2.0 (2 ± 3)
Birth weight (gram) 3087.5
(2816.25 ± 3538.8)*
4500
(3787.5 ± 4765.5)
3607.5
(3306.3 ± 3701.3)
Fetal Sex
Boys
Girls
0.75
0.25
0.8
0.2
0.5
0.5 (Median ± interquartile range (IQR)). Fetal sex is shown as a percentage of the total number of inclusions. The
data was compared using a Kruskal-Wallis test, followed by a Mann-Whitney U test . *=p<0.05.
18
3.2 Memory T cells
3.2.1 CD4+ memory cells
Percentages of CD4+ memory cells
were compared between nulliparous,
primiparous and multiparous women.
Figure 1 shows the proportion
CD4+CD45RO+ cells of CD4+ cells
found in the decidua parietalis and
decidua basalis. No significant
differences were found between the
groups.
CD4+ central memory cells
Central memory (CM) cells express
the lymph node homing receptor CCR7 (CD45RO+CCR7+)(32). There was no significant
difference in proportions CM cells between the three groups (Fig. 6A).
Figure 6. CD4+ memory cells in nulliparous, primiparous and multiparous women. CM cells, CD45RO+CCR7+
(A); EM cells, CD45RO+CCR7-(B); and RM cells, CD45RO+CD103+ (C) as a proportion of CD4+ cells. A
Kruskal-Wallis test was performed (p<0.05). Horizontal lines indicate means.
CD4+ CM cells
%C
D4
+C
D4
5R
O+
CC
R7
+/C
D4
+
bas par bas par bas par
0
10
20
30
40
50
NP PP MP
A CD4+ EM cells
%C
D4
+C
D4
5R
O+
CC
R7
-/C
D4
+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
B
CD4+ TRM cells
%C
D4
+C
D4
5R
O+
CD
10
3+
/CD
4+
bas par bas par bas par
0
5
10
15
20
NP PP MP
C
Figure 1. Proportions CD4+ memory T of CD4+ cells in decidua
basalis and parietalis of nulliparous, primiparous and multiparous
women. A Kruskal-Wallis test was performed (p<0.05). Horizontal
lines indicate means.
CD4+ memory cells
%C
D4
+C
D4
5R
O+
/CD
4+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
bas
par
19
CD4+ EM cells
Effector memory cells lack the surface marker CCR7(32). There was no significant difference
in the proportions of effector memory cells with the phenotype CD4+CD45RO+CCR7-
between NP, PP and MP women (Fig. 6B).
CD4+ TRM cells
Resident memory cells are characterized by the cell surface marker CD103(32). There was no
significant difference in proportion CD4+CD45RO+CD103+ cells of CD4+ cells between
NP, PP and MP women (Fig. 6C).
3.2.2 Activated CD4+ Memory T Cells
Following activation, T cells express the cell surface marker CD69(35). Within the different
memory cell subsets, the proportions of activated cells were determined by comparing the
cells that stained positive for CD69 to the original population. However, no significant
differences were found between the groups for CD4+ memory cells, CM cells, EM cells or
RM cells (Fig. 7).
Figure 7. CD4+ activated memory cells in nulliparous, primiparous and multiparous women. Proportions
activated CD4+ memory cells of CD4+ memory cells(A); proportions activated CM cells of CM cells
(CD45RO+CCR7+CD69+) (B); proportions activated EM cells of EM cells (CD45RO+CCR7-CD69+)(C);
proportions activated RM cells of RM cells (CD45RO+CD103+CD69+)(D). A Kruskal-Wallis test was
performed (p<0.05). Horizontal lines indicate means.
Activated CD4+ memory cells
%C
D4
+C
D4
5R
O+
CD
69
+/C
D4
+C
D4
5R
O+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
A Activated CD4+ CM cells
%C
D4
+C
D4
5R
O+
CC
R7
+C
D6
9+
/
CD
45
RO
+C
CR
7+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
B
Activated CD4+ EM cells
%C
D4
+C
D4
5R
O+
CC
R7
-CD
69
+/
CD
45
RO
+C
CR
7-
bas par bas par bas par
0
10
20
30
40
50
NP PP MP
C Actvivated CD4+ TRM cells
%C
D4
+C
D4
5R
O+
CD
10
3+
CD
69
+/
CD
45
RO
+C
D1
03
+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
D
20
3.2.3 CD8+ Memory T cells
CD8+ Memory cells
The CD8+ memory cell subsets were
determined using the same markers and
compared in the same way as CD4+ memory
cell subsets. No differences in percentage
CD8+ memory cells with a CD45RO+
phenotype were seen between the groups
(Fig. 8).
CD8+ CM cells
The percentage of CCR7+ CM cells
within the CD8+CD45RO+ population
was determined for NP, PP and MP
women. No significant differences were found between the groups (Fig. 9A).
Figure 9. CD8+ memory cells in nulliparous, primiparous and multiparous women. CM cells, CD45RO+CCR7+
(A); EM cells, CD45RO+CCR7-(B); and RM cells, CD45RO+CD103+ (C) as a proportion of CD4+ cells. A
Kruskal-Wallis test was performed, followed by a Mann-Whitney U test (#=p<0.1). Horizontal lines indicate
means.
CD8+ CM cells
%C
D8
+C
D4
5R
O+
CC
R7
+/C
D8
+
bas par bas par bas par
0
5
10
15
20
NP PP MP
A CD8+ EM cells
%C
D8
+C
D4
5R
O+
CC
R7
-/C
D8
+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
B#
CD8+ TRM cells
%C
D8
+C
D4
5R
O+
CD
10
3+
/CD
8+
bas par bas par bas par
0
20
40
60
NP PP MP
C
Figure 8. CD8+ memory T cells in nulliparous, primiparous and
multiparous women. A Kruskal-Wallis test was performed
(p<0.05). Horizontal lines indicate means.
CD8+ memory cells
%C
D8
+C
D4
5R
O+
/CD
8+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
21
CD8+ EM cells
We found a trend of higher levels of effector memory cells with a CD8+CD45RO+CCR7-
phenotype cells in the decidua parietalis of NP women compared to PP women (p<0.1)(Fig.
9B).
CD8+ RM cells
There were no significant differences in CD8+CD45RO+CD103+ RM cell proportions of
CD8+ cells between NP, PP and MP women (Fig. 9C).
3.2.4 Activated CD8+ memory T cells
Activation in the CD8+ memory cell population activation was also examined using the
activation marker CD69. No significant differences were found between the groups for CD8+
memory cells, CM cells, EM cells or RM cells (Fig. 10).
Figure 10. CD8+ activated memory cells in nulliparous, primiparous and multiparous women. Proportions
activated CD8+ memory cells of CD8+ memory cells(A); proportions activated CM cells of CM cells
(CD45RO+CCR7+CD69+) (B); proportions activated EM cells of EM cells (CD45RO+CCR7-CD69+)(C);
proportions activated RM cells of RM cells (CD45RO+CD103+CD69+)(D). A Kruskal-Wallis test was
performed (p<0.05). Horizontal lines indicate means.
Activated CD8+ EM cells
%C
D8
+C
D4
5R
O+
CD
69
+/C
D4
5R
O+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
A Activated CD8+ CM cells
%C
D8
+C
D4
5R
O+
CC
R7
+C
D6
9+
/
CD
45
RO
+C
CR
7+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
B
CD8Temactivated
%C
D8
+C
D4
5R
O+
CC
R7
-CD
69
+/
CD
45
RO
+C
CR
7-
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
C Activated CD8+ RM
%C
D8
+C
D4
5R
O+
CD
10
3+
CD
69
+/
CD
45
RO
+C
D1
03
+
bas par bas par bas par
0
20
40
60
80
100
NP PP MP
D
22
3.3 Tregs
Regulatory T cells were identified by the combination of positive staining for the cell surface
marker Foxp3+ and negative staining for the cell surface marker CD127(35). The Treg cells
were only analysed in decidua basalis tissue because of the small amount of cells that could
be isolated from decidua parietalis tissue.
3.3.1 CD4+ Treg cells
The proportions of cells with a CD4+Foxp3+CD127- phenotype of CD4+ cells in decidua
basalis tissue were compared between NP, PP and MP women. No significant differences
were found between the groups (Fig. 11A).
Activated CD4+ Treg cells
Expression of HLA-DR indicates activation of the Treg cell(35). The proportion activated
Tregs of CD4+ cells was significantly lower (p<0.05) in the NP group compared to the PP
and MP group (Fig. 11B).
Suppressive CD4+ Treg cells
The immune-suppressive molecule CTLA-4 is expressed on antigen-experienced cells(35).
There were no significant differences in proportions CD4+Foxp3+CD127-CTLA-4+ cells of
CD4+ cells between the three groups (Fig. 11C).
Figure 11. CD4+ Tregs in decidua basalis tissue. Proportions Tregs (A), activated Tregs (B) and suppressive
Tregs (C) of CD4+ cells for NP, PP and MP women. A Kruskal-Wallis test was performed followed by a Mann-
Whitney U test (*=p<0.05). Horizontal lines indicate means.
CD4+ Treg cells
%C
D4
+F
oxp
3+
CD
12
7-/
CD
4+
np ppm
p
0
5
10
15
20
25
A Activated CD4+ Treg cells
%C
D4
+F
oxp
3+
CD
12
7-H
LA
-DR
+/C
D4
+
np ppm
p
0
20
40
60
80
100
B
*
Suppressive CD4+ Treg cells
%C
D4
+F
oxp
3+
CD
12
7-C
TL
A4
+/C
D4
+
np ppm
p
0
20
40
60
80
100
C
23
3.3.2 CD4+ Memory Tregs
CD4+ memory Treg cells
Memory Treg cells were identified by their surface marker CD45RO(35). Between the three
groups there was no significant difference in percentage of CD4+Foxp3+CD127-CD45RO+
memory Tregs (Fig. 12A).
Activated CD4+ memory Treg cells
There was no difference in levels of memory Tregs with the phenotype CD4+Foxp3+CD127-
CD45RO+HLA-DR+ between the three groups. However, comparison of the NP to the MP
group using a Mann-Whitney U test, showed significantly lower levels of activated memory
Tregs in the NP group (p<0.05) (Fig. 12B).
Suppressive CD4+ memory Treg cells
No difference was found in levels of suppressive CD4+ memory Treg cells between the three
groups. However, when comparing the NP group to the PP and MP group combined, using a
Mann-Whitney U test, the proportion suppressive CD4+ memory Treg cells of CD4+ cells in
the NP group was significantly lower (p<0.05) (Fig. 12C).
Figure 12. CD4+ memory Tregs in decidua parietalis tissue. Proportions memory Tregs of CD4+ cells (A),
proportions activated memory Tregs of memory Tregs (B) and proportions suppressive memory Tregs of
memory Tregs (C) for NP, PP and MP women. A Kruskal-Wallis test was performed followed by a Mann-
Whitney U ((*)= p<0.05 for comparison of NP to PP/MP). Horizontal lines indicate means.
CD4+ memory Treg cells
%C
D4
+F
oxp
3+
CD
12
7-C
D4
5R
O+
/CD
4+
np ppm
p
50
60
70
80
90
100
A Activated CD4+ memory Treg cells
%C
D4
+F
oxp
3+
CD
12
7-C
D4
5R
O+
HL
A-D
R+
/
CD
4+
Fo
xp
3+
CD
12
7-C
D4
5R
O+
np ppm
p
0
10
20
30
40
B
( )*
Suppressive CD4+ memory Treg cells
%C
D4
+F
oxp
3+
CD
12
7-C
D4
5R
O+
CT
LA
4+
/
CD
4+
Fo
xp
3+
CD
12
7-C
D4
5R
O+
np ppm
p
0
20
40
60
80
100
C
( )*
24
3.3.3 CD8+ Treg cells
Proportions cells with a CD8+Foxp3+CD127- phenotype of CD8+ cells in decidua parietalis
tissue were compared between NP, PP and MP women. No significant differences were found
between the groups (Fig. 13A).
Activated CD8+ Treg cells
There was no significant difference in proportions of CD8+ activated Treg cells between the
three groups (Fig. 13B).
Suppressive CD8+ Treg cells
We found a trend of higher levels of suppressive CD8+ Tregs with a CD8+Foxp3+CD127-
CTLA-4+ phenotype in MP women compared to NP women (p<0.1)(Fig. 13C). When
comparing only the NP group to the MP group using a Mann-Whitney U test, the proportion
of suppressive CD8+ Treg cells was significantly lower in the NP group (p<0.05).
Figure 13. CD8+ Tregs in decidua parietalis tissue. Proportions Tregs (A), activated Tregs (B) and suppressive
Tregs (C) of CD8+ cells for NP, PP and MP women. A Kruskal-Wallis test was performed followed by a Mann-
Whitney U ((*)= p<0.05 for comparison of NP to PP/MP). Horizontal lines indicate means.
CD8+ Treg cells
%C
D8
+F
oxp
3+
CD
12
7-/
CD
8+
np ppm
p
0
5
10
15
20
A Activated CD8+ Treg%
CD
8+
Fo
xp
3+
CD
12
7-H
LA
-DR
+/C
D8
+
np ppm
p
0
10
20
30
40
50
B
Suppressive CD8+ Treg cells
%C
D8
+F
oxp
3+
CD
12
7-C
TL
A4
+/C
D8
+
np ppm
p
0
20
40
60
80
100
C
( )*
25
3.3.4 CD8+ memory Tregs
There were no significant differences in levels of CD4+Foxp3+CD127-CD45RO+ memory
Tregs between the three groups (Fig. 14A).
Activated CD8+ memory Treg cells
There were no significant differences in levels of activated memory Tregs with the phenotype
CD8+Foxp3+CD127-CD45RO+HLA-DR+ between the three groups (Fig. 14B).
Suppressive CD8+ memory Treg cells
No differences were found in levels of suppressive CD8+ memory Treg cells between the
three groups. However, when comparing only the NP to the MP group using a Mann-Whitney
U test, levels of suppressive memory Tregs were significantly lower in the NP group
(p<0.05)(Fig. 14C).
Figure 14. CD8+ memory Tregs in decidua parietalis tissue. Proportions memory Tregs of CD8+ cells (A),
proportions activated memory Tregs of memory Tregs (B) and proportions suppressive memory Tregs of
memory Tregs(C) for NP, PP and MP women. A Kruskal-Wallis test was performed followed by a Mann-
Whitney U ((*)= p<0.05 for comparison of NP to PP/MP). Horizontal lines indicate means.
CD8+ memory Treg cells
%C
D8
+F
oxp
3+
CD
12
7-C
D4
5R
O+
/CD
8+
np ppm
p
0
20
40
60
80
A Activated CD8+ memory Treg cells%
CD
8+
Fo
xp
3+
CD
12
7-C
D4
5R
O+
HL
A-D
R/
CD
8+
Fo
xp
3+
CD
12
7-C
D4
5R
O+
np ppm
p
0
10
20
30
40
50
B
Suppressive CD8+ memory Treg cells
%C
D8
+F
oxp
3+
CD
12
7-C
D4
5R
O+
CT
LA
4+
/
CD
8+
Fo
xp
3+
CD
12
7+
CD
45
RO
+
np ppm
p
0
20
40
60
80
100
C
*
26
4. Discussion
In this study we examined the effect of parity on the subsets and characteristics of memory T
cells in decidual tissue. The effect of parity on memory T cell subsets was tested by
comparing lymphocytes extracted from decidual tissue of nulliparous, primiparous and
multiparous women. Additionally we determined and compared regulatory T cell subsets.
Our results show no differences in memory cell subsets between the three groups.
Interestingly parity does seem to have an effect on the Treg cell population. We found
significantly higher proportions of CD4+ and CD8+ Tregs with an activated and suppressive
phenotype as well as memory Tregs in primiparous and multiparous women compared to
nulliparous women.
4.1 Memory T cells
Contrary to what we hypothesized, we did not find higher proportions memory T cells or
memory T cells subsets in primiparous and multiparous women compared to nulliparous
women. There could be several reasons why we did not find higher levels of memory cells in
multiparous women:
Previous research showed that during pregnancy, maternal CD8+ T-lymphocytes can be
primed by fetal antigens and both lymphocytes and fetal antigens persist in the maternal blood
post-partum(15,38,39). A study of Barton et al. studied these antigen-experienced cells in a
mouse model performing re-exposure to fetal antigens by means of a subsequent
pregnancy(40). They observed no enhanced expansion of fetal-primed CD8+ memory cells
during pregnancy in parous mice. Our findings appear to be in line with these data, as we also
did not find higher levels of CD8+ memory T cells in the decidua of parous women. Barton et
al. found that pregnancy in mice induces T cells with a dysfunctional effector phenotype that
exhibit impaired cytokine production(40). During a subsequent pregnancy, these T cells
exhibited limited proliferative capacity. The limited proliferative capacities of the pregnancy
induced memory T cells could be an explanation for the lack of higher proportions of those
memory cells in multiparous women. More research examining proliferative capacities of
human memory T cells isolated from pregnant women in cell proliferation assays, could
provide very useful information.
A study by Kieffer et al. found significantly higher proportions of CD4+ and CD8+ effector-
memory T cells, CD4+ central-memory cells and activated CD4+ memory T cells in the
peripheral blood of non-pregnant multiparous women compared to nulligravid women. Our
results do not show increased levels of memory T cell subsets in the decidua of primiparous
or multiparous women. There could be several reasons why parity seems to increase the levels
of memory T cells in the peripheral blood, and not in the decidua:
the placenta features several specific characteristics which constrain a T cell mediated
response against fetal antigens. At the fetal-maternal interface, where fetal trophoblast cells
are directly exposed to the maternal immune system, large scale antigen recognition by
maternal T cells is not possible because trophoblast cells do not express classic MHC
molecules which are essential for direct antigen-recognition(1,41). Only indirect antigen
recognition mediated by maternal antigen-presenting cells enables maternal T cells to detect
the alloantigens(14,40).The expression of IDO by trophoblast cells also constrains an attack
of the maternal immune system because IDO inhibits rapid proliferation of T cells(23,42).
Trophoblast cells also express Fas ligand, which induces apoptosis in Fas-expressing
27
activated CD4+ and CD8+ T lymphocytes(23,43). Finally, complement activation which
promotes a pro-inflammatory immune response is inhibited by expression of the Crry-protein
on trophoblast cells(23,44).
All together these mechanisms minimise a T cell mediated immune response against the
allogeneic fetus at the fetal-maternal interface. Perhaps these mechanisms also prevent an
influx of memory T cells that reside in the peripheral blood, into the decidua during a second
pregnancy(45). This would explain why higher proportions memory T cells are seen in the
peripheral blood of multiparous women but do not correspond with higher proportions
memory T cells in the decidua of multiparous women.
Another explanation for the relatively equal levels of memory cells in the decidua of
nulliparous, primiparous and multiparous women could be that memory cells migrate near the
end of pregnancy. During pregnancy, levels of T cells are increased in the decidua compared
to the peripheral blood, but post-partum increased levels of memory T cells are found in the
peripheral blood(36,38). Possibly, memory T cell migrate from the decidua towards the
peripheral blood at the end of pregnancy where they reside until antigen re-exposure during
the next pregnancy.
Alternatively, memory T cells subsets could diminish in the third trimester under influence of
hormonal changes. Progesterone is up regulated in the third trimester and has been shown to
suppress the T cell response, especially CD4+ effector activity(46).
Both of these explanations suggest that analysis of term decidua tissue will not show
differences in memory T cell subsets. Perhaps a difference can be found in decidua tissue in
the first or second trimester, however this is obviously difficult in healthy human pregnancies.
To our knowledge this is the first study to compare memory T cells in decidua tissue between
nulliparous, primiparous and multiparous women. Therefore we cannot compare our findings
to other studies.
4.2 Tregs
We found higher levels of activated CD4+ Tregs in multiparous and primiparous women, and
higher levels of CD8+CTLA-4+ memory Treg cells in multiparous women compared to
nulliparous women. When comparing only the nulliparous to the multiparous group
additionally higher levels of activated CD4+ memory Tregs, CD4+CTLA-4+ memory Tregs
and CD8+CTLA-4+ Tregs were found in multiparous women.
Regulatory T cells are potent suppressive immune regulators and are extensively studied for
their role in pregnancy. Previous research described an accumulation of CD4+ Tregs in the
decidua as well as antigen-independent systemic accumulation during pregnancy(25,30)
4.2.1 Memory Tregs
Memory T cells are antigen experienced cells that rapidly proliferate upon re-exposure to an
antigen and thereby protect against harmful pathogens during a subsequent exposure.
Regulatory T cells are also able to memorise antigens, these antigen experienced Tregs can
suppress a T cell response against a specific antigen after re-exposure(31,35). Memory Tregs
are probably very useful in establishing a rapid tolerance for the fetus in a subsequent
pregnancy(28,31).
28
In this study, we found higher levels of memory Tregs with an activated phenotype in
primiparous and multiparous women compared to nulliparous women (Fig. 12B, Fig. 12C,
Fig. 14C). These data could support the hypothesis that memory Tregs are important in
establishing immunological tolerance in a subsequent pregnancy.
Our findings are in line with a mouse study by Rowe et al. that showed fetal-antigen specific
Tregs persisted at higher levels post-partum and rapidly re-expanded during a subsequent
pregnancy in the peripheral blood of mice(31). Interestingly these mice had a lower risk of
pregnancy complications in their second pregnancy compared to mice in which Tregs were
inhibited. As we previously mentioned, in humans the risk of pre-eclampsia is higher in a first
pregnancy. Furthermore, lower percentages of Tregs are seen in pre-eclamptic women(29).
Our data support the importance of Tregs in healthy pregnancies and show that multiparous
women exhibit higher percentages of Tregs in the decidua. Taken all together, perhaps
memory Tregs could be involved in the reason why the risk of pre-eclampsia is lower in
secondary pregnancies. Comparing the memory Treg population in the decidua of healthy
women to pre-eclamptic women could provide more insight into the role of memory Tregs in
pre-eclampsia.
4.2.2 Suppressive Tregs
We found an increased percentages of CD4+ and CD8+ Tregs as well as CD4+ and CD8+
memory Tregs that express CTLA-4 in multiparous women compared to primiparous women
(Fig. 12C, 13C, 14C). CTLA-4 is a marker of suppressive function which is up regulated in
Tregs after activation by antigens(47).
The significantly higher levels of Tregs expressing CTLA-4 we found in multiparous women,
indicate a subset of Tregs with higher functional capacities. This could explain why we did
not find higher levels of memory T cells in multiparous women as we hypothesized. CTLA-
4+ Tregs can attenuate antigen-specific T cell responses by competitive binding of co-
stimulatory molecules which are necessary for antigen-specific activation of T cells(48,49).
Also CTLA-4 has been shown to induce IDO expression on APC’s which, as we mentioned
previously, inhibits rapid T cell proliferation(50–52)
Therefore we propose that in multiparous women, the abundance of CTLA-4+ Tregs could
suppress the expansion of memory T cells that were formed during a previous pregnancy.
However, more research investigating the functional capacities of decidual Tregs is needed.
The importance of CTLA-4+ Tregs in fetal-maternal immune tolerance can be illustrated by
research that showed that CTLA-4 is down regulated on decidual Tregs in miscarriage
compared to normal pregnancy and in spontaneous abortion compared to induced
abortion(53).
4.2.3 Activated Tregs
The cell surface marker HLA-DR is a late activation marker. However, HLA-DR+ Tregs were
also identified as a distinct Treg subset with a high suppressive function(54). Our results show
higher levels of activated CD4+ Tregs and CD4+ memory Tregs in primiparous and
multiparous women compared to nulliparous women(Fig. 11B, Fig. 12B). A decrease of
HLA-DR+ expression on Tregs was seen in the peripheral blood of women in preterm
labour(55). Perhaps these high levels of HLA-DR+ Tregs are involved in mechanisms that
protect multiparous women against immunological pregnancy complications. However as
29
mentioned above, more research investigating the functional capacities of decidual Tregs is
needed.
There is no definitive consent on phenotypical characterization of Tregs. In this study Foxp3
and low CD127 expression were used to define Tregs. CD25Bright
is also a commonly used
marker for CD4+ Treg cells in decidual tissue. CD4+CD25+ T cell subsets show regulatory
capacities, however CD25 is also expressed on effector T cells(13,15,16). CD25 therefore is
not an exclusive Treg cell marker. Foxp3+ was found to be expressed on cells with regulatory
function within the CD25+ as well as the CD25- T cell subsets(15,17,18). Foxp3 is now
considered to be a more specific marker for regulatory T cells.
As shown in mouse models, CD127 is not down regulated in all Tregs, which would make
CD127- an unreliable marker for Treg characterization. However, we have used CD127- as an
additional marker next to Foxp3. The combination of these two markers has been shown to
specifically identify the Treg cell population(56).
Tregs in decidual tissue have been intensively studied for their role in immune tolerance for
the fetus, the majority of this research has used CD25+ for phenotypic characterization of
Tregs. This marker was not included in our research as we believe the use of Foxp3 is more
accurate. This should be kept in mind when comparing our findings to earlier research on
Tregs.
4.3 Strengths and limitations
Strengthening this study, we analysed memory T cell subsets at the site of the immunological
challenge, the fetal-maternal interface. The cells present in the decidua are directly involved
in the immune response towards the fetus and therefore analysis of these subsets show us
which cells play an important role in the regulation of the immune response towards the fetus.
Furthermore, we included the placentas of women who gave birth by an elective caesarean
section. During labour the release of hormones, such as progesterone, and contractions
influence lymphocyte subsets(46). The delivery of the baby by a caesarean section minimises
the influence of labour on the lymphocytes. Also the protocol of isolation of the lymphocytes
from the placenta was started immediately after delivery, which minimised the time for
lymphocytes to change or deteriorate once oxygen supply is cut off.
Another strength of this study are the extensive antibody panels that were used to determine
the memory T cell and Treg cell subsets. Within the memory cell panel, the central-memory
cell population, effector-memory cell population and resident memory cell population could
be determined as well as the proportions of activated cells within these population. The Treg
cell panel included several markers for Tregs and additional suppression and activation
markers.
Several limitations should also be considered. First of all, due to the small number of elective
caesarean sections in the study period, only a small number of placenta’s were available for
this study. Therefore these results cannot be assumed to be representative for the general
population, however they give an indication of what could be expected if more patients were
included in this research. Also because the groups are relatively small, the data show a high
dispersion. Statistical analysis can therefore only show large differences. If more patients
were to be included, statistical significant differences between the groups could be examined
with greater reliability and precision.
Also, because of the small number of patients available for this research, we did not exclude
patients based on their BMI even though BMI is known to influence the immune
30
response(57). However, only the BMI of the women in the nulliparous group was statistically
significantly different from the other groups, no statistical significant difference was found
between the primiparous and multiparous group.
Furthermore we analysed lymphocytes that were extracted from term decidual tissue. The
lymphocyte populations present in decidual tissue are known to change dynamically during
pregnancy. This study only allows conclusions about the proportions of different cell subsets
present at the end of pregnancy, but these might be different in earlier stages of pregnancy.
Isolation of lymphocytes from decidual tissue is more complicated than from peripheral
blood, and the amount of cells obtained is much lower. This limits possibilities for analysis of
these cells and prohibited us from assessing the functional features of these cells.
4.4 Conclusion
In conclusion, the results of this study show that parity did not affect memory T cell subsets in
the decidua as we did not find differences in memory T cell levels between the groups.
However, parity did seem to affect regulatory T cell subsets in the decidua. We found higher
levels of CD4+ and CD8+ Treg and memory Treg cells in primiparous and multiparous
women compared to nulliparous women. Furthermore, we found higher levels of antigen
experienced Tregs with a suppressive phenotype in primiparous and multiparous women
compared to nulliparous women.
This study contributes to existing knowledge on the development of immunological tolerance
for the fetus by showing that parity affects regulatory T cells population at the fetal-maternal
interface. Whether these alterations in the regulatory T cells population at the fetal-maternal
interface are involved in lowering the risk of immune associated complications of pregnancy
remains to be investigated.
4.5 Future perspectives
To assess the role of memory T cells and Tregs in maternal-fetal immune tolerance, more
research is required. First, more patients should be included in this study for more powerful
and reliable statistical analysis. Also studying the migration patterns of memory T cells
circulating in the peripheral blood could provide useful insight in the role of memory T cell in
maternal fetal immune tolerance. Next to the migration patterns of memory cells, the
proliferative and functional capacities of regulatory T cells isolated from human decidua
tissue could provide more insight into the role of regulatory T cells in immunological
tolerance for the allogeneic fetus.
31
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35
Acknowledgements
First of all, I would like to thank my supervisors dr. Marijke Faas and dr. Jelmer Prins for
making this project possible. Especially I would like to thank dr. Prins and Tom Kieffer for
their help and guidance, and for always being available to answer our questions.
For all the long hours in the lab together, for your positive and patient attitude and for the fun
we have had, I would like to thank Anne Laskewitz. I would not have wanted to do this
project without you.
Also I would like to thank Bart de Haan for his guidance in the laboratory as well as the
immunology and endocrinology research group.
Finally, I would like to thank all the patients included in this research for their participation.
36
Abstract Nederlands
Introductie. Tijdens de zwangerschap bestaat er een unieke situatie waarbij het moederlijk
immuunsysteem de semi-allogene foetus niet aanvalt. Hoe deze tolerantie ontstaat is nog niet
volledig opgehelderd, maar een aangepaste reactie van T-lymfocyten speelt zeker een rol. Uit
onderzoek blijkt dat er tijdens de zwangerschap T-geheugen cellen specifiek voor het foetale
antigeen worden aangemaakt. Ook is bekend dat een aantal immunologische
zwangerschapscomplicaties vaker voorkomen bij de eerste zwangerschap. Dit onderzoek
heeft tot doel de invloed van pariteit op de verschillende T-geheugen cel en de regulatoire T-
cel populaties in de placenta te bepalen.
Materiaal en methode. Lymfocyten zijn enzymatisch geïsoleerd uit de decidua van nulliparae
(n=4), primiparae (n=5) en multiparae (n=6). De lymfocyten zijn gekleurd met antilichamen
en geanalyseerd door middel van flow cytometrie.
Resultaten. We vonden geen verschil in de geheugen T-cel populaties tussen de drie groepen.
Significant hogere proporties geactiveerde en suppressieve regulatoire T-cellen waren te zien
in primiparae en multiparae. Daarnaast vonden we ook significant hogere proporties
regulatoire T-geheugencellen in primi- en multiparae.
Conclusie. In conclusie hebben we geen effect van pariteit op geheugen T-cel populaties
gevonden. Het doormaken van meerdere zwangerschappen lijkt te zorgen voor een meer
geactiveerde en suppressieve regulatoire T-cel populatie in de decidua en daarnaast te zorgen
voor hogere percentages regulatoire T-geheugen cellen. Verder onderzoek is nodig om te
bepalen wat het effect van deze veranderingen is op het ontwikkelen van immunologische
zwangerschapscomplicaties.
37
Appendix
Protocol Enzymatic isolation of lymphocytes from decidua tissue
Date:
Preparation prior to experiment: Reserve laminar airflow cabinet
Bring Accutase to room temperature
PBS
RPMI
Antibodies
Fc block + mouse serum
Fix/Perm and Perm wash buffer
Basket and PBS to collect the placenta
Percoll solution
40% → 13.2 ml SIP + 19.8 ml RPMI
45% → 18.6 ml SIP + 22.7 ml PBS
68% → 22.4 ml SIP + 10,6 ml RPM
Isolation protocol
Preheat waterbath, collect: scissors, forceps, measuring cup, petri dish, filter, cell- scraper,
pipette, 50ml tubes, gloves, PBS, accutase, filters and C-tubes. Turn on radio.
Cut maternal side of decidua basalis, avoid damaged (white, yellow) tissue.
Cut the superfluous villi of the decidua basalis, keep the tissue in immersed in PBS while
preparing.
Remove the amniotic membrane from the chorion and cut the chorion from the basal plate.
Use the cellscraper to separate the decidua parietalis from the chorion, keep the tissue
immersed in PBS while preparing.
Take a sample of basalis and parietalis tissue and tranfser to 0.5ml RNAlater for parietalis and
1 ml RNAlater for basalis.
Transfer tissue to 50ml tubes and add PBS. Mince the tissue using scissors.
Centrifugeer 5 minutes at 1000 rpm. Remove supernatant.
Transfer tissue to C-tubes at 5 ml per tube and resuspend in 10 ml accutase at room
temperature.
Put the C-tubes in the GentleMacs dissociator.
For basalis choose: m-heart 02.01 (17 sec 668 rpr)
For parietalis choose: h-tumor 02 2x (37 sec 235 rpr)
Put the C-tubes in the waterbath and incubate for 45
minutes at 37°C while gently shaking.
After incubation, add 10 ml PBS to ach tube.
Filtrate over a 70 µm Smart Strainer in a 50 ml tubes.
Centrifuge 5 minutes at 1500 rpm at room temperature
Remove supernatant and resuspend in 10 ml RPMI.
Add 10 ml of 40% Percoll solution and resuspend.
Add 10 ml of 68% Percoll solution to a new 50 ml tubes. Slowly layer 12.5 ml of the 45%
solution on top of the 68% Percoll solution. Keep the tube at a 45º angle while layering. Then
layer 20 ml of the 40% Percoll / cell solution on top of the 45% layer in the same way.
Centrifuge for 30 minutes at 2000 rpm without brake.
Take of layer A, then collect the macrophage layer using a 1 ml pipette and transfer to a 50
ml tube. Next, collect the lymphocyte layer using a 1 ml pipette and transfer to a 50 ml tube.
38
Add up PBS to 50 ml and centrifuge for 5 minutes at 1500 rpm at room temperature.
Resuspend in 1 ml of PBS
Count cells using the coulter counter.
Protocol Flow cytometry
Wells needed for experiment:
14 wells
6 lymphocytes
4 macrophages
4 single stains
Preparations before staining
Make 500 µl 1% Fc block + 10% mouse serum:
50µl per well
Add 445µl Facs buffer + 5µl Fcblock + 50µl mouse serum
Make 1200µl BD Fix/Perm working solution 1:4 (keep cold)
200 µl per well
300µl Fix/perm + 900µl diluent
Make 4,8 ml Perm/Wash buffer 1:5 (keep cold)
800µl per well
960µl of Perm/Wash buffer + 3840µl demi
Make FVS
10 µl FVS + 490 µl PBS
Cell preparation Add 1.000.000 cells per well in 96 well plates according to scheme, 3.000.000 cells for
macrophages
Spin down at 1800 RPM for 3min (RT) and discard supernatant
Stain with 50ul FVS for 15 min at RT in the dark
Wash twice with PBS (2min, 1800, RT).
Block cells with 50 µl 1% FC block + 10 % mouse serum for 10 minutes (RT)
Spin down at 1800 RPM for 3 min (4C) and discard supernatant
Add 50µl extracellular antibodymix to all wells according to scheme
Incubate on ice in the dark for 30 min.
Spin down at 1800 RPM for 3 min (4C) and discard supernatant
Wash 2X with 200µl PBS
Fixating cells
Fix all samples in 200 µl BD fix/perm in the fridge for 40 min.
Spin down at 1800 RPM for 3 min(4C) and discard supernatant
Wash 2x with 200 µl Perm wash buffer
Resuspend macrophages in 150 µl PBS and keep dark in the fridge until analysis
Only for Tregs & Tmem
Add 50 µl of intracellular antibodymix to all wells according to scheme
Incubate on ice in the dark for 30min
Wash 2x with 200 µl Perm wash buffer
Resuspend the samples in 150 µl PBS
Keep cells in the dark in the fridge until analysis
39
Staining protocol
1
2
3
4
5
6
7 8 9 10 11 12
A par Tregs Tmem Cntrl ss FVS
B
C Macro Cntrl ss FVS
D
E bas Tregs Tmem Cntrl ss FVS
F
G Macro Cntrl ss FVS
H
Cell count
Basalis lymfo Basalis macro
1ml 1ml
1E6 1E6
Parietalis lymfo Parietalis macro
1ml 1ml
1E6 1E6
Antibody Panels Extracellulair
Tregs
AB Intra/extra Verdunning Volume (x2) Kanaal
CD4 Extra 1:100 1 µl APC-H7
CD8 Extra 1:200 0.5 µl Pe-Cy7
CD45RO Extra 1:20 5 µl V500-C
CD127 Extra 1:20 5 µl PE
HLA-DR Extra 1:200 0.5 µl APC
PBS 88 µl
Tmem
AB Intra/extra Verdunning Volume
(2x)
kanaal
CD4 Extra 1:100 1 µl APC-H7
CD8 Extra 1:200 0.5 µl Pe-Cy7
CD45RO Extra 1:20 5 µl V500-C
CD103 Extra 1:50 2 µl BV421
CCR7 Extra 1:10 10 µl PE
CD69 Extra 1:20 5 µl APC
PBS 76.5 µl
40
Macrophages
AB Extra Verdunning Volume
(2x)
Kanaal
HLA-DR Extra 1:10 10 µl APC
CD163 Extra 1:50 2 µl BV421
CD14 Extra 1:20 5 µl V500-C
CD209 Extra 1:20 5 µl FITC
ICAM3 Extra 1:50 2 µl PE
CD80 Extra 1:20 5 µl Pe-Cy7
CD45 Extra 1:20 5 µl APC-H7
PBS 66 µl
Intracellulair
Treg
AB Intra/extra Verdunning Volume (x2) Kanaal
CTLA-4 Intra 1:20 5 µl BV421
FOXP3 Intra 1:20 5 µl AF488
PBS 90 µl
Tmem
AB Intra/extra Verdunning Volume
(2x)
kanaal
FOXP3 Intra 1:20 5 µl AF488
PBS 95 µl
Beads
Vortex beads (30 seconds).
Divide 1 drop over 1 tubes (30 µl per tube ).
Add 0.5 µl antibody.
Incubate for 30 minutes in the fridge.
Wash 2 times with 200 µl PBS (5min, 1800 rpm) and remove supernatant using a pipette tip.
Resuspend in 150 µl PBS, keep cold in the dark until analysis.