Iodine-Deficient BiO1.2I0.6 Coupling with Bi2O3 for Degradation of Volatile Organic Compounds under Simulated Sunlight Irradiation

Article abstract of DOI:10.1002/cssc.201901627

A photocatalyst with a high separation efficiency of carriers is crucial for the photocatalytic efficient decomposition of volatile organic compounds (VOCs). In this work, a new iodine-deficient BiO_1.2I_0.6/Bi₂O₃ photocatalyst was developed through a solvothermal approach coupled with thermal decomposition. The 2 wt % BiO_1.2I_0.6/Bi₂O₃ sample demonstrated optimal photocatalytic activity for the degradation of toluene, achieving a 55.1 % mineralization ratio under 8 h of irradiation, which was 14.9 and 2.1 times higher than those of BiO_1.2I_0.6 and Bi₂O₃, respectively. Compared with BiO_1.2I_0.6 and Bi₂O₃, 2 wt % BiO_1.2I_0.6/Bi₂O₃ exhibited the weakest fluorescence band, highest photocurrent density, and lowest impedance; thus suggesting that Bi₂O₃ compounded by 2 wt % BiO_1.2I_0.6 distinctly promoted the separation efficiency of carriers. The hydroxyl radical (^.OH) was the main active species, apart from holes, for the photocatalytic degradation of toluene.

Full text of DOI:10.1002/cssc.201901627

Received: 19 November 2018ꢀ ꢀ Revised: 31 January 2019ꢀ ꢀ Accepted: 5 February 2019
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DOI: 10.1111/aji.13106
O R I G I N A L A R T I C L E
Bidirectional regulation between 1st trimester HTR8/SVneo
trophoblast cells and in vitro differentiated Th17/Treg cells
suggest a fetal‐maternal regulatory loop in human pregnancy
Songcun Wangꢀ| Jinfeng Qianꢀ| Fengrun Sunꢀ| Mengdie Liꢀ| Jiangfeng Yeꢀ|
Mingqing Liꢀ| Meirong Du ꢀ| Dajin Li
Laboratory for Reproductive Immunology,
NHC Key Lab of Reproduction Regulation
Problem: During normal pregnancy, delicate crosstalk is established between fetus‐
(Shanghai Institute of Planned Parenthood
Research), Shanghai Key Laboratory of
Female Reproductive Endocrine Related
derived trophoblasts and maternal immune cells to ensure maternal‐fetal tolerance
and successful placentation. Dysfunction in these interactions has been highly linked
Diseases, Hospital of Obstetrics and
Gynecology, Fudan University Shanghai
Medical College, Shanghai, China
to certain pregnancy complications.
Method of study: Naïve CD4+T cells were cultivated with or without 1st trimester
derived trophoblast cell line HTR8/SVneo cells in the absence or presence of T helper
Correspondence
Meirong Du and Dajin Li, Laboratory for
Reproductive Immunology, NHC Key Lab of
Reproduction Regulation (Shanghai Institute
of Planned Parenthood Research), Shanghai
Key Laboratory of Female Reproductive
Endocrine Related Diseases, Hospital of
Obstetrics and Gynecology, Fudan University
17 (Th17) or regulatory (Treg)cell‐inducing differentiation conditions. After 5 days of
co‐culture, HTR8/SVneo cells and CD4+T cells were harvested and analyzed using
flow cytometry.
Results: CD4+T cells exposed to HTR8/SVneo cells showed enhanced induction of
CD4+Foxp3+Treg cells with strong expression of TGF‐β1 and inhibitory molecules
Shanghai Medical College, Shanghai, China.
Emails: dmrlq1973@sina.cn; djli@shmu.edu.cn
(cytotoxic T lymphocyte‐associated protein‐4 [CTLA‐4], T‐cell immunoglobulin
mucin‐3 [Tim‐3], and programmed cell death‐1 [PD‐1]). Though not effecting Th17
Funding information
This work was supported by grant from the
differentiation, exposure to HTR8/SVneo cells promoted increased expression of
proliferative and apoptotic markers on Th17 cells. Co‐culture with Th0 cells, or dif‐
ferentiated Th17 or Treg cells, down‐regulated Caspase‐3 and MMP‐9 (but not
MMP‐2) expression in HTR8/SVneo cells, while promoting Ki67 expression.
Conclusions: HTR8/SVneo cells regulated maternal CD4+T‐cell differentiation, re‐
sulting in the expansion of immunosuppressive Treg cells, while CD4+T cells might
promote the growth, and control the invasiveness of HTR8/SVneo cells. Thus, a bidi‐
rectional regulatory loop might exist between trophoblasts and maternal immune cell
subsets, thereby promoting harmonious maternal‐fetal crosstalk.
Nature Science Foundation from National
Nature Science Foundation of China (NSFC)
(31700799 to SCW, 81630036, 91542116
and 31570920 to MRD), the National Basic
Research Program of China (2015CB943300
to DJL and MRD), the National Key R&D
Program of China (2017YFC1001403 to DJL
and MRD), the Program of Shanghai Academic/
Technology Research Leader (17XD1400900
to MRD), the Innovation‐oriented Science
and Technology Grant from NHC Key Lab of
Reproduction Regulation (CX2017‐2 to MRD),
the Shanghai Sailing Program (17YF1411600 to
SCW), the Training Program for Young Talents
of Shanghai Health System (2018YQ07 to
SCW), Shanghai Chenguang Program (18CG09
to SCW), Development Fund of Shanghai
Talents (2018110 to SCW) and the Introduction
Project of Suzhou Clinical Medicine Expert
Team (grant SZYJTD201708 to DJL).
K E Y W O R D S
HTR8/SVneo cells, pregnancy, Th17 cells, Treg cells, trophoblasts
Songcun Wang and Jingfeng Qian contributed equally to this manuscript.
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made
© 2019 The Authors American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd
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wileyonlinelibrary.com/journal/aji ꢀ ꢀ
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1ꢀ|ꢀINTRODUCTION
2ꢀ|ꢀMATERIALS AND METHODS
2.1ꢀ|ꢀHuman samples
The establishment of maternal‐fetal tolerance and successful pla‐
centation are key events during early pregnancy. Under normal
conditions, the maternal immune system accepts the semi‐alloge‐
neic fetus, which protects both mother and fetus against infection.
Disruption of this immune balance, however, causes the placenta
and fetus to be attacked as a foreign organ transplant, resulting in
pregnancy failure.1 Extravillous trophoblasts (EVT), the main cell
type involved in the placentation process, invade the underlying de‐
cidua, dissolve the extracellular matrix (ECM), and migrate into the
uterine spiral arteriolar walls, remodeling the uterine vasculature.
Inadequate EVT invasion has been closely associated with several
pregnancy‐associated diseases, including recurrent pregnancy loss
(RPL), pre‐eclampsia (PE), and gestational trophoblastic diseases.2
T‐cell subsets, especially CD4+ helper T (Th) cells, play a pivotal
role in successful pregnancy.3 Driven by a set of transcriptional reg‐
ulators and cytokines, naive CD4+T cells are able to differentiate into
distinct subsets, including Th1, Th2, Th17, and Treg cells.4 A polar‐
ization toward Th2 bias in the maternal immune response has long
been considered the main mechanism of tolerance induction toward
the fetus.5 According to recent studies, the balance between reg‐
ulatory (Treg) and T helper 17 (Th17) cells is also important in the
maintenance of normal pregnancy, whereas the shift in the Th17/
Treg ratio toward Th17 cells has been proposed as a cause for sev‐
eral pregnancy‐associated diseases, such as RPL, PE, and gestational
diabetes mellitus.6‐8
This study was approved by the Human Research Ethics Committee
of the Obstetrics and Gynecology Hospital, Fudan University. All
participants signed a written informed consent form. Heparinized
peripheral blood was obtained from pregnant women who had one
or more previous normal pregnancies without any miscarriage, ex‐
cluding those diagnosed with endocrine diseases, tumor, infection,
etc (n = 18, mean age [years]: 29.7 ± 0.6, range: 25‐35). Samples
were immediately collected for isolation of peripheral blood mono‐
nuclear cells (PBMCs).
2.2ꢀ|ꢀIsolation of human cells
PBMCs were isolated from peripheral blood samples of pregnant
women using Ficoll (Huajing, China) density gradient centrifuga‐
tion. Naïve CD4+T cells were isolated through magnetic affinity
cell sorting using the Naïve CD4+T Isolation Kit II (MiltenyiBiotec,
Germany). An antibody cocktail (biotin‐conjugated monoclonal anti‐
bodies against CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36,
CD45RO, CD56, CD123, TCRγ/δ, HLA‐DR, and Glycophorin A) and
anti‐biotin microbeads were added to PBMCs following the manu‐
facturer's instructions. The suspension of cells (naïve CD4+T cells)
was recovered in a new tube, while magnetically labeled unwanted
cells remained bound to the original tube through the magnetic
field. The obtained cells were fluorescently stained for CD45RA
and CD4. Approximately 3 × 106 naïve CD4+T cells with a purity of
around 99% were obtained from each woman. Cells were exten‐
sively washed and resuspended in RPMI 1640 (HyClone, USA) sup‐
plemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/
mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL amphotericin B
(Sangon Biotech, China).
Decidual immune cells (DICs) not only regulate the maternal
immune response to promote fetal semi‐allograft tolerance but
also mediate the implantation and trophoblast invasion.9,10 At the
same time, trophoblasts mediate interactions between the fetus and
mother for the exchange of nutrients, gases, waste products, as well
as for the regulation of immune tolerance.11 EVTs are potential can‐
didates for educating maternal immune cells to generate a tolerant
microenvironment at the maternal‐fetal interface. At present, stud‐
ies regarding the interaction between trophoblasts and DICs have
shown that trophoblast has the unique ability of instructing DICs to
develop a regulatory phenotype for fetal tolerance.12‐15 However,
the regulatory effect of trophoblasts on CD4+T‐cell differentiation,
especially on Th17/Treg differentiation, remains poorly understood.
Knowledge regarding the influence of Th17/Treg differentiation on
the biological behaviors of trophoblasts is also limited.
2.3ꢀ|ꢀIn vitro differentiation of Th17 and Treg cells
Naïve CD4 T cells were cultured in precoated plates with anti‐
CD3 (5 μg/mL, Clone OKT‐3, BioLegend, USA) + anti‐CD28 (1 μg/
mL, Clone CD28.2，BioLegend, USA) antibodies and maintained in
media supplemented with IL‐2 (10 ng/mL, PeproTech, USA) (Group
Th0). Recombinant IL‐6 (50 ng/mL, PeproTech, USA), recombinant
TGF‐β1 (10 ng/mL, PeproTech, USA), anti‐IFN‐γ mAb (10 μg/mL,
Clone B27, BioLegend, USA), and anti‐IL‐4 mAb (10 μg/mL, Clone
8D4‐8, BioLegend, USA) were added to generate Th17 cells (Group
Th17). Recombinant TGF‐β1 (50 ng/mL, PeproTech, USA), anti‐IL‐6
mAb (10 μg/mL, Clone MQ2‐13A5, BioLegend, USA), anti‐IFN‐γ
mAb (10 μg/mL, Clone B27, BioLegend, USA), and anti‐IL‐4 mAb
(10 μg/mL, Clone 8D4‐8, BioLegend, USA) were added to generate
Treg cells (Group Treg). Media were changed every 48 hours. This
protocol was based on previous publications.17,18 For intracellular
cytokine analysis, brefeldin A (10 mg/mL, BioLegend, USA), phor‐
bol 12‐myrstate 13‐acetate (PMA)(50 ng/mL, BioLegend, USA),
Based on the aforementioned observations, we assumed that
trophoblasts might affect Th17/Treg cell differentiation from naive
CD4+T cells, leading to the induction of Treg cell expansion at the
maternal‐fetal interface. In turn, differentiated CD4+T cells might af‐
fect the biological functions of trophoblasts. Based on this hypoth‐
esis, we investigated the effect of trophoblasts on Th17/Treg cell
differentiation from naive CD4+T cells and trophoblast phenotypic
markers of function modulated by Th17/Treg cells generated in vitro
using the immortalized human first‐trimester EVT cell line HTR8/
SVneo,16 which has been widely used as a substitute for human pri‐
mary trophoblasts.

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and ionomycin (1 μg/mL, BioLegend, USA) were added 4 hours be‐
fore the end of the culture. The cells were then harvested, stained,
and analyzed through flow cytometry. After 5 days of culture,
8.3 ± 1.7% of CD4+IL‐17A+ cells and 13.2 ± 1.7% of CD4+Foxp3+
cells were obtained.
that showed a significant difference with Kruskal‐Wallis test. A
P value of <0.05 was considered significant. For variables with a
P value less than 0.05 following ANOVA, the post hoc Dunnett t
test was performed to determine differences between each group.
All analyses were carried out using the GraphPad Prism 5 software
(GraphPad, San Diego, CA).
2.4ꢀ|ꢀCo‐culture of CD4+T and HTR8/SVneo cells
3ꢀ|ꢀRESULTS
HTR8/SVneo cells (2 × 105) were initially cultured in 6‐well flat‐bot‐
tom plates in RPMI 1640 medium supplemented with 10% FBS the
day before co‐culture with naïve CD4+T cells. When the adherent
HTR8/SVneo cells reached 50%, 4 × 105 Th0, Th17, and Treg cells
(according to the different differentiation agents mentioned previ‐
ously) were seeded on the top of the HTR8/SVneo layer for 5 days
in media comprising different differentiation agents. After induction,
the number of CD4+T cells in each well was approximately 2 × 106.
While in the co‐culture system, the cell number was approximately
double in this amount. To exclude the effect of HTR8/SVneo cell
proliferation on CD4+T cells, HTR8/SVneo cells were pre‐treated
with mitomycin C for 30 min (10 mM) in some wells. For intracellular
cytokine analysis of CD4+T cells, brefeldin A (10 mg/mL, BioLegend,
USA), PMA (50 ng/mL, BioLegend, USA), and ionomycin (1 μg/mL,
BioLegend, USA) were added 4 hours before the end of the culture.
HTR8/SVneo cells and CD4+T cells were then harvested, stained,
and analyzed through flow cytometry. After culture, the proportion
of live cells (assessed by the 7‐AAD− cells) was around 90%.
3.1ꢀ|ꢀTrophoblasts contribute to Treg cell
differentiation from maternal Naïve CD4+T cells
To examine whether trophoblasts influence the differentiation
of Th17/Treg cells, Naïve CD4+T cells from PBMCs of pregnant
women were cultivated with or without human EVT cell line HTR‐8/
SVneo cells in the absence or presence of Th17‐/Treg‐inducing dif‐
ferentiation conditions (described in the Methods section). As de‐
picted in Figure 1A, A mix of differentiated Th17 cells (8.3 ± 1.7% of
CD4+IL‐17A+ cells) and non‐differentiated CD4+T cells was obtained
under Th17 induction. IL‐17 expression in Treg‐polarized cells was
approximately 1.7 ± 0.2% (Figure S1). HTR8/SVneo cells had no ef‐
fect on Th17 cell differentiation as the proportion of CD4+IL‐17A+
cells did not vary after co‐culture with HTR8 cells.
Treg cells were differentiated as described in the Methods sec‐
tion. As shown in Figure 1B,C, 13.2 ± 1.7% of differentiated Treg
cells (CD4+Foxp3+ cells) were obtained accompanied by up‐regula‐
tion of IL‐10 and TGF‐β1 expression. The proportion of CD4+Foxp3+
cells and the expression of TGF‐β1 increased after co‐culture with
HTR8/SVneo cells. Foxp3 expression in Th17‐polarized cells was ap‐
proximately 3.5 ± 0.2% (Figure S1). To exclude the effect of HTR8/
SVneo cell proliferation on CD4+T cells, we used mitomycin C to in‐
hibit the proliferation of HTR8/SVneo cells and found that HTR8/
SVneo cell proliferation did not affect CD4+T‐cell differentiation
(Figure S2). These results support the fact that HTR8/SVneo cells
increase the frequency of Treg cells after in vitro differentiation.
2.5ꢀ|ꢀFlow cytometry
Cell surface molecular expression and intracellular cytokine produc‐
tion were evaluated using flow cytometry. AlexaeFluor® 488‐con‐
jugated anti‐human Foxp3 or MMP‐9; FITC‐conjugated anti‐human
CD4; PE‐conjugated anti‐human CD45RA, MMP‐2, T‐cell immu‐
noglobulin mucin‐3 (Tim‐3), or TGF‐β1; PE/CY7‐conjugated anti‐
human IL‐10, CD45 or IL‐17A; PerCP‐conjugated anti‐human CD4;
AlexaeFluor® 647‐conjugated anti‐human Caspase‐3, APC‐con‐
jugated anti‐human CTLA‐4; Brilliant Violet 421‐conjugated anti‐
human Ki‐67 or IL‐17a; Brilliant Violet 510‐conjugated anti‐human
CD45 or PD‐1 antibodies (BioLegend, USA) were used. Details re‐
garding Ab are presented in Table S1. For intracellular staining, cells
were fixed and permeabilized using the Fix/Perm kit (BioLegend,
USA). Flow cytometry was performed on a Beckman‐Coulter CyAn
ADP cytometer and analyzed with FlowJo software (Tree Star, USA).
3.2ꢀ|ꢀTrophoblasts regulate the function of
differentiated Th17/Treg cells
To directly assess whether HTR8/SVneo cells regulated the bio‐
logical functions of Th17/Treg cells generated in vitro, CD4+T‐cell
apoptosis and proliferation were analyzed. As shown in Figure 2A,
apoptosis (assessed by the expression of Caspase‐3) of generated
Th17 cells was higher than that of Th0 cells but lower than that of
Th17 cells co‐cultured with HTR8/SVneo cells. Meanwhile, Treg cell
apoptosis decreased after co‐culture with HTR8/SVneo cells. HTR8/
SVneo cells also increased the proliferation (assessed by the expres‐
sion of Ki‐67) of Th17 cell, but had no effect on Treg cell proliferation
(Figure 2B). Taken together, these data indicated that HTR8/SVneo
cells promoted the renewal of Th17 cells and inhibited the apoptosis
of Treg cells.
2.6ꢀ|ꢀStatistical analysis
Normally distributed variables were presented as means and SD.
ANOVA was used to evaluate differences in normally distributed
variables with homogeneity of variance among groups. Variables
with skewed distribution were described using median and inter‐
quartile range. The Kruskal‐wallis test was used to assess the dif‐
ference in variables with skewed distribution among groups. The
Bonferroni multiple comparisons test was performed for variables
Inhibitory receptors on immune cells, such as CTLA‐4, Tim‐3,
and PD‐1 regulate the T cell–mediated immune response and have

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F I G U R E 1 ꢀ HTR8/SVneo cells contributed to the differentiation of Treg cells from maternal naïve CD4+T cells. (A) Maternal naïve CD4+T
cells were differentiated in the presence of IL‐2 (Th0) or IL‐2 + TGF‐β1 + IL‐6 + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Th17) for 5 days. In some
wells, CD4+T cells were co‐cultured with HTR8/SVneo cells. IL‐17A expression in T cells was analyzed through flow cytometry. (B and C)
Maternal naïve CD4+T cells were differentiated in the presence of IL‐2 (Th0) or IL‐2 + TGF‐β1 + anti‐IL‐6 mAb + anti‐IFN‐γ mAb + anti‐
IL‐4 mAb (Treg) for 5 days. In some wells, CD4+T cells were co‐cultured with HTR8/SVneo cells. Foxp‐3 expression and IL‐10 and TGF‐β1
production in CD4+T cells were analyzed through flow cytometry. **P < 0.01, ***P < 0.001, compared to the Th0 group. ##P < 0.01,
###P < 0.001, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are representative of three
independent experiments
been proposed as functional markers of specific T‐cell subsets.19,20
Next, we analyzed CTLA‐4, Tim‐3, and PD‐1 expression in in vi‐
tro‐generated Th17/Treg cells cultivated with or without HTR8 cells.
As shown in Figure 3, compared to Th0 cells, Th17 cells expressed
higher levels of Tim‐3 and PD‐1, while Treg cells expressed more
CTLA‐4, Tim‐3, and PD‐1. HTR8/SVneo cells increased the expres‐
sion of all inhibitory receptors in Treg cells, but had no influence on
their expression in Th17 cells.
with Th0, Th17, or Treg cell, HTR8/SVneo cell apoptosis (assessed
using Caspaase‐3+ cells) was inhibited, while HTR8/SVneo cell prolif‐
eration (assessed using the Ki‐67+ cells) was promoted (Figure 4A,B).
MMP‐2 and MMP‐9 are two important gelatinases involved in ECM
remodeling during trophoblast invasion.21 The effects of CD4+T cells
on MMP‐2 and MMP‐9 expression were examined using flow cytom‐
etry. As shown in Figure 4C,D, CD4+T cells decreased MMP‐9 produc‐
tion in HTR8/SVneo cells, but had no effect on MMP‐2 expression.
3.3ꢀ|ꢀCD4+T cells modulate the biological
behaviors of trophoblast
4ꢀ|ꢀDISCUSSION
We then studied whether in vitro‐generated Th17/Treg cells regu‐
lated the biological behaviors of HTR8/SVneo cells. After co‐culture
A successful pregnancy relies on the maternal immune system's
tolerance toward the semi‐allogeneic fetus and sufficient placental

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F I G U R E 2 ꢀ HTR8/SVneo cells regulated the apoptosis and proliferation of differentiated T cells. Maternal naïve CD4+T cells were
differentiated in the presence of IL‐2 (Th0), IL‐2 + TGF‐β1 + IL‐6 + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Th17) or IL‐2 + TGF‐β1 + anti‐IL‐6
mAb + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Treg) for 5 days. In some wells, T cells were co‐cultured with HTR8/SVneo cells. Caspase‐3 (A) and
Ki‐67 (B) expression in CD4+T cells was analyzed through flow cytometry.*P < 0.05, ***P < 0.001, compared to Th0 group. ###P < 0.001,
compared to Th17 group. ^P < 0.05, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are
representative of three independent experiments
F I G U R E 3 ꢀ HTR8/SVneo cells promoted the expression of inhibitory receptors in differentiated Treg cells. Maternal naïve CD4+T cells
were differentiated in the presence of Th0‐, Th17‐, or Treg‐inducing differentiation agents for 5 days. In some wells, CD4+T cells were
co‐cultured with HTR8/SVneo cells. Cytotoxic T lymphocyte‐associated protein‐4 (CTLA‐4) (A), Tim‐3 (B), and programmed cell death‐1
(PD‐1) (C) expression in CD4+T cells was analyzed through flow cytometry. **P < 0.01, ***P < 0.001, compared to Th0 group. #P < 0.05,
###P < 0.001, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are representative of three
independent experiments
formation. As such, a complex and reciprocal interaction between
the maternal immune system and fetal trophoblasts is required to
maintain normal pregnancy. The present study utilized the well‐
characterized human first‐trimester EVT cell line HTR8/SVneo to
demonstrate the notable role of these cells in regulating CD4+T‐cell
differentiation, resulting in the expansion of immunosuppressive
Treg cells. Meanwhile, CD4+T cells also modulated the biological
functions of HTR8/SVneo cells. To our knowledge, this study pre‐
sented novel findings suggesting a unique maternal‐fetal co‐culture
system could be used to regulate the Th17/Treg ratio and that a
positive regulatory loop might exist between trophoblasts and ma‐
ternal immune cell subsets, promoting the harmonious maternal‐
fetal crosstalk.
Upon encountering antigens presented by antigen presenting
cells or being driven by a set of cytokines, naive CD4+T cells are able
to differentiate into distinct subsets, including Th1, Th2, Th17, and
Treg cells.22 Th2 bias at the maternal‐fetal interface has long been
considered as the main mechanism of tolerance induction toward
the fetus.5 Th17 cells play a critical role in inducing of inflamma‐
tion, while abnormal Th17 cell levels have been associated with the

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F I G U R E 4 ꢀ Differentiated CD4+T cells modulated the biological functions of HTR8/SVneo cells. HTR8/SVneo cells were co‐cultured with
Th0, Th17 and Treg cells (with the indicated agents) for 5 days. Caspase‐3 (A), Ki‐67 (B), MMP‐2 (C) and MMP‐9 (D) expression in HTR8/
SVneo cells was analyzed through flow cytometry.***P < 0.001, compared to the Ctrl group. Data are represented as the mean ± SD. Flow
cytometry plots are representative of three independent experiments
pathogenesis of autoimmune diseases.23 Treg cells express anti‐in‐
flammatory cytokines such as IL‐10 and TGF‐β1, which dampen an
excessively effective immune response.24 Studies have proposed
that alterations in the balance between Th17 and Treg cells may be
associated with the development of RSA.25‐27
Moreover, PD‐1 and CTLA‐4 are critical in the suppressive activity
of Treg cells,32,33 while PD‐1+Tim‐3+Treg cells exhibit higher effec‐
tor function.34 HTR8/SVneo cells, as well as primary trophoblasts
but not decidual stromal cells (data not shown) or OVC1 (an ovarian
cancer cell line), up‐regulated CTLA‐4, Tim‐3, and PD‐1 expression
in Treg cells along with TGF‐β1 production, indicating that fetal‐de‐
rived trophoblasts were pivotal inducers of maternal immune adap‐
tation. HLA‐G, which is selectively expressed in cytotrophoblasts
and invasive EVTs and also expressed in HTR8/SVneo cells,35,36 was
responsible for the training function of Trophblast (data not shown).
Moreover, given that HTR8/SVneo cells could promote CD4+T‐cell
activation (assessed through CD69 expression, data not shown), we
determined that HTR8/SVneo cells at least partially instructed the
CD4+T‐cell phenotype by activating these cells through HLA‐G.
With its high proliferative ability, invasive properties, and ca‐
pacity to avoid immune attack, the human placenta has been re‐
garded as a pseudo‐malignant structure in which a subpopulation of
placental trophoblasts erode the uterus, its underlying stroma and
local blood vessels to achieve adequate maternal‐fetal exchange
of molecules. However, unlike tissue invasion by malignant tumor
cells, trophoblast invasion to the uterus is temporally and locally
controlled by hormones, cytokines, and functional cells at the ma‐
ternal‐fetal interface.34 Accordingly, inadequate EVT growth and
invasion have been closely associated with several pregnancy‐as‐
sociated diseases.2 Like tumor invasion, MMPs are major regulators
of tissue remodeling during pregnancy, with gelatinases MMP‐2 and
MMP‐9 being involved in trophoblast invasion to the spiral arteries.
In cultured trophoblasts, suppression of MMP‐9 expression inhibits
their invasive capability.37 After co‐culture with Th0 cells, or differ‐
entiated Th17 or Treg cells, Caspase‐3 and MMP‐9 (but not MMP‐2)
expression in HTR8/SVneo cells was down‐regulated, while Ki67
Although an appropriate pro‐inflammatory response is
a
prerequisite for successful implantation and defense against
pathogens, multiple regulatory mechanisms are also required to
balance maternal immune cell‐trophoblast interaction through‐
out gestation, which will ultimately determine the fate of preg‐
nancy. Specifically, the fine balance between Th17 and Treg cells
mediates maternal tolerance to the fetus.7 In the present study,
though HTR8/SVneo cells promoted Th17 cell proliferation，they
also up‐regulated the expression of apoptotic marker in Th17 cells.
HTR8/SVneo cell‐conditioned CD4+T cells showed enhanced in‐
duction of the CD4+Foxp3+Treg population with potent TGF‐β1
production and inhibited Treg cell apoptosis, suggesting that the
interaction between EVTs and immune cells might contribute to
the in situ generation of a set of tolerogenic Treg cells and regu‐
late the Th17/Treg ratio to help maintain normal pregnancy. While
Svensson et al have shown that trophoblasts induce functionally
suppressive Treg cells,15 the present study used an alternative pro‐
cedure wherein direct cellular interactions and the ability to fur‐
ther polarize Treg cells were evaluated. Of course, further in vitro
experiments using primary trophoblasts and in vivo functional and
pathological studies are needed to understand the interaction be‐
tween trophoblasts and maternal CD4+T cells.
Inhibitory molecules such as CTLA‐4, Tim‐3, or PD‐1, play a role
in regulating Th17 and Treg cell function to maintain a balanced im‐
mune response.28‐31 For example, Tim‐3 has been associated with a
shift in the balance from Th17 and Treg cells to Treg dominance.30

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expression was promoted, suggesting that CD4+T cells promoted
the growth of trophoblasts and controlled the invasiveness of
human trophoblasts. MMP‐2 is the main gelatinase involved in the
function of early first‐trimester trophoblast (6‐8 weeks). In the late
first trimester (8‐12 weeks), however, both MMP‐2 and MMP‐9 par‐
ticipate in trophoblast invasion.38 Accordingly, our findings show
that CD4+T cells down‐regulated MMP‐9 but not MMP‐2 expres‐
sion in HTR8/SVneo cells. As such, the interaction between CD4+T
cells and trophoblast might play a role in the prevention of exces‐
sive trophoblast invasion in the late first trimester of pregnancy.
Though Th17 cells had been reported to participate in preg‐
nancy‐related pathologies,39 our study and others40 showed that
Th17 cells played important roles in regulating trophoblast func‐
tion. Recent findings have also indicated that not all Th17 cells are
pro‐inflammatory given that Th17 subpopulations with diverse
functions may exist.41 More investigations are needed to explore
whether non‐pathogenic Th17 subsets and pro‐inflammatory Th17
subpopulations co‐exist at the maternal‐fetal interface, which
subsets are involved in the functional regulation of trophoblast,
and which mechanisms are used to modulate Th17 subsets during
pregnancy.
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Questions remain regarding the validity of using immortalized
cell lines to represent the in vivo environment,15,42 though HTR8/
SVneo cells have been proven effective for recapitulating key as‐
pects of EVTs.43 Nonetheless, the interaction between primary
trophoblasts and naïve CD4+T cells and the related mechanisms still
require further study. Moreover, further ELISA assay and flow cy‐
tometry sorting technology would be needed to determine cytokine
production by different T‐cell subsets. Despite these limitations, the
results presented in the present study undoubtedly demonstrated
that trophoblasts might influence the differentiation of naïve CD4+T
cells, while CD4+T cells could in turn modulate the biological func‐
tions of trophoblasts, resulting in Treg expansion and adequate tro‐
phoblast invasion during pregnancy.
12. Du MR, Guo PF, Piao HL, et al. Embryonic trophoblasts induce de‐
cidual regulatory T cell differentiation and maternal‐fetal tolerance
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13. Wang SC, Li YH, Piao HL, et al. PD‐1 and Tim‐3 pathways are asso‐
ciated with regulatory CD8+ T‐cell function in decidua and mainte‐
nance of normal pregnancy. Cell Death Dis. 2015;6:e1738.
14. Wang S, Zhu X, Xu Y, et al. Programmed cell death‐1 (PD‐1) and
T‐cell immunoglobulin mucin‐3 (Tim‐3) regulate CD4+ T cells to in‐
duce Type 2 helper T cell (Th2) bias at the maternal‐fetal interface.
Hum Reprod. 2016;31(4):700‐711.
15. Svensson‐Arvelund J, Mehta RB, Lindau R, et al. The human fetal
placenta promotes tolerance against the semiallogeneic fetus by
inducing regulatory T cells and homeostatic M2 macrophages. J
Immunol. 2015;194(4):1534‐1544.
16. Graham CH, Hawley TS, Hawley RC, et al. Establishment and char‐
acterization of first trimester human trophoblast cells with ex‐
tended lifespan. Exp Cell Res. 1993;206(2):204‐211.
17. Sekiya T, Kashiwagi I, Yoshida R, et al. Nr4a receptors are essential
for thymic regulatory T cell development and immune homeostasis.
Nat Immunol. 2013;14(3):230‐237.
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Mol Biol. 2016;1344:183‐191.
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diseases. Nat Rev Immunol. 2018;18(2):91‐104.
CONFLICT OF INTEREST
The authors disclose no conflict of interests.
ORCID
Meirong Du
Dajin Li
https://orcid.org/0000‐0001‐9998‐1379
https://orcid.org/0000‐0003‐2280‐9727
20. Kudo M. Immuno‐oncology in hepatocellular carcinoma: 2017 up‐
date. Oncology‐Basel. 2017;93(Suppl 1):147‐159.
21. Zhang H, Hou L, Li CM, Zhang WY. The chemokine CXCL6 re‐
stricts human trophoblast cell migration and invasion by sup‐
pressing MMP‐2 activity in the first trimester. Hum Reprod.
2013;28(9):2350‐2362.
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on Th17 cells: function and regulation of human T helper 17 cells in
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.ꢁ
How to cite this article: Wang S, Qian J, Sun F, et al.
Bidirectional regulation between 1st trimester HTR8/SVneo
trophoblast cells and in vitro differentiated Th17/Treg cells
suggest a fetal‐maternal regulatory loop in human pregnancy.
Am J Reprod Immunol. 2019;81:e13106. https://doi.
org/10.1111/aji.13106