Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell–Trophoblast Interactions

Abstract

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

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Citation: Mikhailova, V.; Grebenkina,
P.; Khokhlova, E.; Davydova, A.;
Salloum, Z.; Tyshchuk, E.; Zagainova,
V.; Markova, K.; Kogan, I.; Selkov, S.;
et al. Pro- and Anti-Inflammatory
Cytokines in the Context of NK
Cell–Trophoblast Interactions. Int. J.
Mol. Sci. 2022,23, 2387. https://
doi.org/10.3390/ijms23042387
Academic Editor: Daniela Novick
Received: 26 January 2022
Accepted: 18 February 2022
Published: 21 February 2022
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4.0/).
International Journal of
Molecular Sciences
Article
Pro- and Anti-Inflammatory Cytokines in the Context of NK
Cell–Trophoblast Interactions
Valentina Mikhailova 1,2, * , Polina Grebenkina 1, Evgeniia Khokhlova 1, Alina Davydova 1, Zeina Salloum 1,
Elizaveta Tyshchuk 1, Valeria Zagainova 3, Kseniia Markova 1, Igor Kogan 3, Sergey Selkov 1,2
and Dmitry Sokolov 1,2
1Department of Immunology and Intercellular Interactions, Federal State Budgetary Scientific Institution,
Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott,
199034 St. Petersburg, Russia; grebenkinap@gmail.com (P.G.); evenka95@gmail.com (E.K.);
alyadavydova@gmail.com (A.D.); corbiepost@yandex.ru (Z.S.); tyshhuk.elizaveta@gmail.com (E.T.);
kl.markova94@gmail.com (K.M.); selkovsa@mail.ru (S.S.); falcojugger@yandex.ru (D.S.)
2Department of Immunology, State Budgetary Educational Institution of Higher Professional Education First
Pavlov State Medical University of St. Petersburg under the Ministry of Healthcare of the Russian Federation,
197022 St. Petersburg, Russia
3
Department of Artificial Reproduction Technologies, Federal State Budgetary Scientific Institution, Research
Institute of Obstetrics, Gynecology, and Reproductology named after D.O. Ott, 199034 St. Petersburg, Russia;
zagaynovav.al.52@mail.ru (V.Z.); ikogan@mail.ru (I.K.)
*Correspondence: mva_spb@mail.ru; Tel.: +7-(812)-323-75-45
Abstract:
During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact
interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the
decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and
change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the
development of reproductive pathology. The aim of this study was to assess the effects of cytokines
on NK cells in the presence of trophoblast cells in an
in vitro
model. We used TNF
α
, IFN
γ
, TGF
β
and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant
women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNF
α
caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNF
α
increased
the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGF
β
, the expression
of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary
cultivation of NK-92 cells in the presence of TGF
β
, their cytotoxicity increased. In the case of adding
TGF
β
to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and
NKp44 by pNK cells was reduced. The differences in the effects of TGF
β
in the model using NK-92
cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid
cells from the mononuclear fraction.
Keywords: NK cells; trophoblast; NK-92; pNK; JEG-3; TNFα; TGFβ; CD56; PBMC
1. Introduction
Natural killer cells (NK cells) are cytotoxic, innate immunity lymphocytes that are
characterized by the presence of the surface receptors CD45 and CD56, as well as the
absence of the linear differentiation receptors CD3, CD14 and CD19 [
1
]. Mature NK cells
predominantly circulate in the peripheral blood (pNK). Resident populations of NK cells
are found in lymphoid organs (spleen, tonsils, lymph nodes, Peyer’s patches of the intestine
and thymus) and non-lymphoid organs (liver, lungs and uterus) [
2
,
3
]. One example of
tissue-resident cells is uterine NK cells. During pregnancy, NK cells accumulate in the
decidual membrane, forming a pool of decidual NK cells (dNKs) [
4
]. Fewer NK cells reside
in the endometrium in non-pregnant women [4].
Int. J. Mol. Sci. 2022,23, 2387. https://doi.org/10.3390/ijms23042387 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022,23, 2387 2 of 18
NK cells express various receptors that regulate cytotoxicity towards trophoblast
cells [
5
,
6
]. The receptors have not yet been clearly defined; however, on the basis of their
expression, it is possible to separate NK cells into cytotoxic or regulatory populations [
7
]. The
cells of the uteroplacental complex form a cytokine microenvironment that can influence the
phenotype and functions of NK cells and regulate their interactions with trophoblast cells.
Extravillous trophoblast cells produce TNF
α
and its receptors [
8
], as well as TGF
β
[
9
].
Under the influence of a conditioned medium from trophoblast cells, peripheral blood
monocytes differentiate into macrophages that are similar in their characteristics to the
macrophages of the decidua and produce TGF
β
[
10
]. Through the secretion of TGF
β
,
decidual macrophages can suppress the cytotoxic activity of decidual NK cells [
11
]. TGF
β
stimulates the acquisition of pNK features that are characteristic of dNK cells [
12
] and also
affects the functions of NK cells [
13
]. In addition, TGF
β
reduces IFN
γ
secretion by NK
cells, blocks signals from the NKG2D and CD244 receptors of NK cells and contributes
to a decrease in the expression of NKp30. As a result, the cytotoxic activity of NK cells
ultimately decreases [14,15].
First-trimester decidua macrophages secrete IL-10, TNF
α
and IFN
γ
[
16
–
19
]. The IL-10
receptor consists of two subunits and is expressed on many cells, including NK cells [
19
]. IL-
10 inhibits the cytotoxic and secretory functions of NK cells [
20
]. The increased production
of IFN
γ
and TNF
α
by macrophages leads to the increased apoptosis of trophoblast cells [
16
].
It has been shown in mice that IFN
γ
stimulates a multidirectional change in inhibitory
receptor expression in pNK cells [21].
In addition to microenvironment cell cytokines, NK cells can directly synthesize cytokines.
It has been shown that CD56+ pNK cells produce IFN
γ
, TNF
α
and TGF
β
in the first trimester
of pregnancy [
22
]. Some populations of dNK cells synthesize TNF
α
[
12
,
23
,
24
], IFN
γ
[
12
,
14
],
TGF
β
[
24
] and IL-10 [
23
,
24
] during the first trimester of physiological pregnancy. An imbal-
ance of pro- and anti-inflammatory cytokines in the uteroplacental complex can lead to the
development of reproductive pathology. For example, IL-10 gene expression in the placenta
is reduced in preeclampsia [
25
], while IFN
γ
secretion is increased [
26
], when compared
to normal pregnancy. The increased production of the cytokines IFN
γ
and TNF
α
by dNK
cells and placental macrophages is associated with the development of miscarriage [
16
,
23
].
Thus, a wide range of cytokines have been described in the uteroplacental complex, yet
there are insufficient data on the effects of cytokines on the phenotype and functional state
of NK cells during contact interaction with trophoblast cells. Therefore, the aim of this
work was to assess the effects of the cytokines TNF
α
, IFN
γ
, TGF
β
and IL-10 on the receptor
profile and cytotoxicity of NK cells in coculture with JEG-3 trophoblast cells.
2. Results
2.1. Influence of Cytokines on the Phenotype of the NK-92 Cell Line in Monoculture and under
Co-Cultivation Conditions with the JEG-3 Cell Line
We used a model system of contact cultivation with trophoblast cells, which we
described previously [
27
]. The phenotype of NK-92 cells was assessed after their cultivation
without (hereinafter referred to as monoculture) and in the presence of trophoblast cells
(hereinafter referred to as coculture).
2.1.1. Effect of TNFαon the Phenotype of the NK-92 Cell Line in Monoculture and under
Co-Cultivation Conditions with the JEG-3 Cell Line
The number of NK-92 cells did not change after the addition of TNF
α
to the mono-
culture (Figure 1). In the monoculture, the addition of TNF
α
resulted in a decrease in the
expression of the CD56 receptor by NK-92 cells compared to cultivation without TNF
α
in
both IL-2+ and IL-2-free media (Figure 2a).

Int. J. Mol. Sci. 2022,23, 2387 3 of 18
Figure 1.
The number of NK-92 cells with phenotypes (
a
) CD56+, (
b
) NKG2A+, (
c
) NKG2C+,
(
d
) NKp30+, (
e
) CD57+, (
f
) KIR2DL1+, (
g
) KIR2DS4+ and (
h
) KIR3DL1+ after culturing in mono-
culture and coculture with JEG-3 cell line, with cytokines TNF
α
, IL-10, IFN
γ
and TGF
β
. NI (no
inducer)—cultured without inducers. Cont (control)—cultured with IL-2. The experiments with
each of the cytokines were conducted separately, twice, with three replicates for each experiment.
Significant differences: *—p< 0.05, **—p< 0.01 and ***—p< 0.001 (Kruskal–Wallis test).

Int. J. Mol. Sci. 2022,23, 2387 4 of 18
Figure 2.
The intensity of (
a
) CD56, (
b
) NKG2A, (
c
) NKG2C, (
d
) NKp30, (
e
) CD57, (
f
) KIR2DL1,
(
g
) KIR2DS4 and (
h
) KIR3DL1 receptor expression by NK-92 cells after culturing in monoculture
and coculture with JEG-3 cell line, with cytokines TNF
α
, IL-10, IFN
γ
and TGF
β
. NI (no inducer)—
cultured without inducers. Cont (control)—cultured with IL-2. The experiments with each of the
cytokines were conducted separately, twice, with three replicates for each experiment. Significant
differences: *—p< 0.05, **—p< 0.01 and ***—p< 0.001 (Kruskal–Wallis test).
In the coculture, compared with the monoculture, the number of NK-92 cells with the
phenotype CD57+, KIR2DL1+, KIR3DL1+ and CD127+ increased in the presence of TNF
α
in the culture medium without IL-2 (Figure 1e,f,h and Figure S1h). In the case of culturing
in a medium with IL-2 and TNF
α
, the number of NKG2A+, CD57+, KIR3DL1+ and CD127+

Int. J. Mol. Sci. 2022,23, 2387 5 of 18
NK-92 cells increased in the coculture compared to the monoculture (Figure 1b,e,h and
Figure S1h).
In the case of the coculture, the addition of TNF
α
led to an increase in the expression of
NKG2A and NKG2C receptors compared to the cell coculture without TNF
α
. These changes
were revealed after cultivation in media either without IL-2 or with IL-2 (Figure 2b,c).
In the presence of TNF
α
, the expression of the receptors CD56, NKG2A, NKG2C,
CD57 and CD122 by NK-92 cells increased in the coculture compared to the monoculture
in media either without IL-2 or with IL-2 (Figure 2a–c,e and Figure S2g).
2.1.2. Effect of IL-10 on the Phenotype of the NK-92 Cell Line in Monoculture and under
Co-Cultivation Conditions with the JEG-3 Cell Line
In the case of using medium without IL-2, the addition of IL-10 to the medium caused
an increase in the number of KIR2DL3+ and CD161+ cells for the NK-92 cell line in the
coculture compared to monoculture (Figure S1c,f). When cultivated in a medium with IL-2
and IL-10, the number of KIR2DL3+ cells of the NK-92 line also increased in the coculture
compared to monoculture (Figure S1c).
The expression of the CD56 and CD57 receptors by NK-92 cells increased in the
coculture compared to monoculture when using IL-10-containing medium either with IL-2
or without it (Figure 2a,e).
2.1.3. Influence of IFNγon the Phenotype of the NK-92 Cell Line in Monoculture and
under Co-Cultivation Conditions with the JEG-3 Cell Line
IFN
γ
caused an increase in the expression of KIR3DL1 by NK cells only in the case of
the monoculture of NK-92 cells in a medium without IL-2 (Figure 2h).
In IFN
γ
-containing media without IL-2 or with IL-2, the number of NK-92 cells
with the KIR2DS4+ and CD161+ phenotype increased in the coculture compared to the
monoculture (Figure 1g and Figure S1f). In the medium with IL-2 and IFN
γ
, we observed
that the number of NK-92 cells with the NKG2A+ and CD215+ phenotype also increased in
the case of the coculture compared to the monoculture (Figure 1b and Figure S1i).
In the medium without IL-2 but with IFN
γ
, the expression of the NKp44 receptor by
NK-92 cells increased in the case of the coculture compared to the monoculture (Figure S2a).
In the presence of IFN
γ
, the expression of CD56, KIR2DS4 and CD127 by NK cells increased
in the coculture compared to the monoculture, when using the medium either without
IL-2 or with IL-2 (Figure 2a,g and Figure S2h). In the medium with IL-2 and IFN
γ
, the
expression of the NKp30 receptor by NK-92 cells was reduced in the case of the coculture
compared to monoculture (Figure 2d).
2.1.4. Effect of TGFβon the Phenotype of the NK-92 Cell Line in Monoculture and under
Co-Cultivation Conditions with the JEG-3 Cell Line
TGF
β
in monoculture reduced the relative number of NK-92 cells with the NKG2A+
KIR2DL4+ phenotype compared to cultivation without TGF
β
, in the case of culturing
NK cells in media both without IL-2 and with IL-2 (Figure 1b and Figure S1b). TGF
β
in
combination with IL-2 in monoculture led to a decrease in the number of NK-92 cells with
the NKp30+ and CD117+ phenotype compared to the monoculture cultured only with
IL-2 (Figure 1d and Figure S1e). TGF
β
in the monoculture increased the intensity of CD56
expression by NK-92 cells and reduced the intensity of NKp30 expression in the media
both without IL-2 and with IL-2 (Figure 2a,d).
The expression of the receptors KIR2DL4, CD161 and CD127 by NK-92 cells increased
in the coculture compared to the monoculture in the presence of TGF
β
in the culture media
both without IL-2 and with IL-2 (Figure S2b,f,h).
The addition of TGFβto the coculture resulted in a decrease in the number of NK-92
cells expressing NKG2A and CD62L compared to the coculture without TGF
β
(Figure 1b
and Figure S1d). These changes were established using culture media both without IL-2
and with IL-2.

Int. J. Mol. Sci. 2022,23, 2387 6 of 18
The number of NK-92 cells with the KIR2DL1+ KIR2DS4+ KIR3DL1+ KIR2DL3+
phenotype increased in the coculture compared to monoculture in medium supplemented
with TGF
β
(Figure 1a–h and Figure S1c). These changes were established using culture
media both without IL-2 and with IL-2.
TGF
β
in medium without IL-2 reduced the intensity of NKp30 expression by NK-92
cells in coculture compared with the coculture without TGF
β
(Figure 2d). In the medium
with IL-2, TGF
β
increased the expression of the CD56 receptor by NK-92 cells in coculture
compared to the coculture without TGF
β
(Figure 2a). The expression of the CD161 receptor
by NK cells increased in the coculture after the addition of TGF
β
, compared to the coculture
without TGFβ, using media both without IL-2 and with IL-2 (Figure S2f).
The results of the assessment of the effects of the cytokines TNF
α
, IL-10, IFN
γ
and
TGFβare shown in Scheme 1.
Scheme 1.
Effect of TNF
α
, IL-10, IFN
γ
and TGF
β
on NK-92 cells in different culturing variants. The
left part of the diagram shows data for the intensity of NK cell expression of receptors (MFI); the right
part of the diagram shows data for the number of NK cells. Four cultivation situations are shown in
each rectangle: NK cells in monoculture (NK), NK cells cultured with IL-2 (NK+IL-2), NK cells in
coculture with JEG-3 trophoblast cells (NK+JEG) and NK cells in coculture with JEG-3 trophoblast
cells cultured with IL-2 (NK+JEG+IL-2). Changes in the expression or relative number of NK cells as
a result of the addition of a cytokine in each culture situation are shown inside the box. Changes in
the expression or number of NK cells in the case of cultivation of cells in coculture compared with
monoculture, in the presence of a cytokine in the culture medium, are shown by arrows to the left
and right of the rectangles.

Int. J. Mol. Sci. 2022,23, 2387 7 of 18
2.2. Cytotoxicity of NK-92 Cell Line after Culture with Cytokines
The death of the target cells increased when NK cells were added to JEG-3 trophoblast
cells compared to the baseline death levels. After the pre-cultivation of NK cells for 24 h in
the presence of TNF
α
and IL-10, an increased cytotoxicity of NK cells was found compared
to the function of inactivated NK cells (Figure 3a). After the pre-culture of NK cells for 96 h
in the presence of TGF
β
, the cytotoxicity of the NK cells was increased compared to the
function of unstimulated NK cells (Figure 3b).
Figure 3.
The NK-92 cells’ cytotoxicity towards JEG-3 cells after 24 h (
a
) and 96 h (
b
) of NK cell
pre-culturing with cytokines TNF
α
, IL-10, IFN
γ
and TGF
β
. Experiments with each cytokine were
conducted separately, three times, using four replicates in each experiment. Significant differences:
*—p< 0.05, **—p< 0.01 and ***—p< 0.001 (Kruskal–Wallis test).
2.3. Phenotype of Peripheral Blood NK Cells in Monoculture and under Co-Cultivation Conditions
with the JEG-3 Cell Line
The analysis of the phenotype of pNK cells before cultivation revealed no differences
between two subgroups of healthy, non-pregnant women. In this regard, all the examined
women were combined into one group to assess the effect of the JEG-3 trophoblast cell
line on pNK cells. Since both the phenotypic profile of NK-92 cells and their cytotoxicity
changed after a 96-h cultivation in the presence of TGF
β
, this cytokine was chosen to
analyze phenotypic changes in a system using peripheral blood NK cells. Figure 4shows
representative graphs for CD56 expression by NK-92 cells and pNK cells.
After the cultivation of peripheral blood mononuclear cells (PBMC) with TGF
β
, the
number of pNK cells with the phenotype NKp44+ and KIR2DS4+ (Figure 5c,d) and the
expression of CD56, KIR2DL1 and KIR2DS4 (Figure 6) were reduced compared to unstimu-
lated pNK cells.

Int. J. Mol. Sci. 2022,23, 2387 8 of 18
Figure 4.
The intensity of CD56 receptor expression by NK-92 cells (
a
,
b
) and peripheral blood NK
cells (
c
,
d
). Cells were cultured without inducers (grey) and with TGF
β
(dark green). Isotype control
is shown in black.
Figure 5.
The number of pNK cells with (
a
) CD56+, (
b
) NKG2C+, (
c
) NKp44+ and (
d
) KIR2DS4+
phenotypes after monoculture without inducers (peripheral blood mononuclear cells (PBMC)) and
with TGF
β
(PBMC + TGF
β
), and in coculture with JEG-3 cells without cytokines (PBMC + JEG-3)
and with TGF
β
(PBMC + JEG-3 + TGF
β
). PBMCs were collected from healthy, non-pregnant women
(n= 21). Significant differences: **—p< 0.01 and ***—p< 0.001 (Wilcoxon matched-pairs rank test).

Int. J. Mol. Sci. 2022,23, 2387 9 of 18
Figure 6.
The intensity of (
a
) CD56, (
b
) KIR2DL1, (
c
) NKp44 and (
d
) KIR2DS4 receptor expression
by pNK cells after monoculture without inducers (PBMC) and with TGF
β
(PBMC + TGF
β
), and
in coculture with JEG-3 cells without cytokines (PBMC + JEG-3) and with TGF
β
(PBMC + JEG-3 +
TGF
β
). PBMCs were collected from healthy, non-pregnant women (n= 21). Significant differences:
***—p< 0.001 (Wilcoxon matched-pairs rank test).
After culturing with JEG-3 trophoblast cells, the number of pNK cells with the NKG2C+
phenotype and the expression of CD56 by pNK cells were reduced compared to the cells
cultivated without trophoblasts (Figure 5a).
After culturing mononuclear cells in the presence of trophoblast cells and TGF
β
, the
number of NKp44+ pNK cells (Figure 5) and the expression of the CD56, KIR2DL1, NKp44
and KIR2DS4 receptors by NK cells were reduced compared to pNK cells in coculture with
JEG-3 trophoblast cells without the addition of TGFβ(Figure 6).
The intensity of CD56 expression by pNK cells was reduced in the case of the culti-
vation of mononuclear cells in a medium containing TGF
β
with JEG-3 trophoblast cells,
compared with a monoculture of PBMCs in a medium with TGFβ(Figure 6).

Int. J. Mol. Sci. 2022,23, 2387 10 of 18
3. Discussion
TNF
α
is a pleiotropic cytokine [
28
]. TNF
α
enhances the activating effect of IL-2 and
also stimulates the cytotoxicity of NK cells against target cells
in vitro
[
29
,
30
]. We showed
that cultivation with TNF
α
reduced the expression of CD56 by NK-92 cells. We also found
that, after pre-cultivation with TNF
α
for 24 h, the cytotoxicity of NK-92 cells against JEG-3
trophoblast cells was enhanced. These changes in cell phenotype and function can be
regarded as the induction of the CD56dim cytotoxic phenotype in the presence of TNFα.
It has been reported that NK cells, after
in vitro
cultivation in the presence of feeder
cells expressing membrane-bound IL-21, can change their phenotype and begin to express
NKG2C and CD57 receptors, retaining their ability to induce cytotoxicity [
31
]. These
changes, together with the acquisition of HLA-DR, are regarded as being characteristic
of adaptive NK cells [
31
]. In women with previous pregnancies, “pregnancy-trained”
decidual NK cells have been described, which are characterized by the increased expression
of NKG2C [
32
]. Endometrial NK cells have been shown to express more NKG2A than
pNK cells [
33
]. In the present work, in the case of culture with JEG-3 trophoblast cells, the
addition of TNF
α
caused the increased expression of the activating receptor NKG2C and
the inhibitory receptor NKG2A. An increased number of CD57+ NK-92 cells and increased
expression of CD57 were also found in the case of coculture compared to monoculture.
These changes in the NK-92 cell line phenotype after exposure to trophoblast cells may
reflect the regulatory effect of trophoblast cells and the induction of a memory-like NK
cell phenotype in the case of pregnancy. TNF
α
is produced by endometrial cells in the
secretory phase of the menstrual cycle [
34
], as well as by dNK cells in the first trimester
of pregnancy [
23
]. In this regard, the effect of TNF
α
in combination with the contact
interaction with trophoblast cells cannot be regarded solely as pro-inflammatory.
At the same time, it is known that the secretion of TNF
α
, as well as IFN
γ
, by dNK
cells is increased in spontaneous miscarriage [
23
]. However, we found that the cytotoxicity
of NK cells against JEG-3 cells after the preliminary cultivation of NK-92 cells for 96 h in
the presence of TNF
α
did not change compared to that for non-activated NK cells. It is
likely that the absence of changes in the cytotoxicity of NK cells against JEG-3 cells after
cultivation with TNF
α
is associated with a short-term effect of the activation of NK-92 cells
and the loss of the activated state after a 96-h incubation.
Another pro-inflammatory NK cell cytokine is IFN
γ
. We found that IFN
γ
caused an
increase in the expression of the activation receptor KIR3DL1 by NK-92 cells in monoculture.
After cultivation in a medium with IFN
γ
, the effect of trophoblast cells on NK-92 cells was
preserved: an increase in the expression of CD56 and KIR2DS4 compared to that in the
monoculture was detected. Song X. et al. described a correlation between the cytotoxic
function of NK-92 cells and their secretion of IFN
γ
upon interaction with K562 target
cells [
35
]. Despite the fact that IFN
γ
is a pro-inflammatory cytokine, we were unable to
detect differences between the cytotoxicity of NK cells cultured with IFN
γ
and the activity
of unstimulated NK cells against JEG-3 cells. This lack of effect could be associated with
both the concentration of IFN
γ
used and the time of cell stimulation. Previously, we found
that, in the case of the short-term cultivation of NK-92 cells and JEG-3 cells, IFN
γ
at a
similar concentration (1000 IU/mL) caused an increase in the cytotoxic activity of NK cells
compared to that of non-activated cells [
36
]. It has been shown that, in the case of the
long-term cultivation (72 h) of JEG-3 cells in the presence of IFN
γ
, the expression of HLA-G
by trophoblast cells is increased [
37
]. Thus, according to our data and previously published
data, the interaction of NK cells and trophoblast cells in the presence of IFN
γ
does not
merely involve contact cytolysis, but also includes changes in the phenotypic characteristics
of both NK cells and trophoblast cells, which can also affect their cytotoxic function.
IL-10 is an immunoregulatory cytokine whose production has been demonstrated in
dNK cells [
38
] and decidual macrophages [
18
]. IL-10 was found to affect the interaction of
dNK cells and the dendritic cells of the uteroplacental complex in a mouse model [
39
]. We
did not establish a direct effect of IL-10 in either the mono- or coculture of the NK-92 and
JEG-3 cell lines. With IL-10 in the culture medium, the expression of CD56 and CD57 in NK

Int. J. Mol. Sci. 2022,23, 2387 11 of 18
cells increased in the coculture compared to the monoculture. These changes in phenotype
indicate the persistent modulating effect of trophoblast cells in the presence of IL-10.
In the current study, we found that after 24 h of preculturing, IL-10 strongly improved
the cytotoxic activity of NK-92 cells towards JEG-3 cells. It was previously established
that, when IL-10 was added to a model system to assess contact cytolysis by NK cells
towards trophoblast cells, it caused an increase in the cytotoxicity of NK-92 cells [
36
]. After
preculturing with IL-10, pNK cells have been shown to possess increased lytic activity
against K562 cells [
40
,
41
] and an increase in granzyme B content [
41
]. In addition, in the
case of cultivation for 24 h, IL-10 has been shown to reduce the expression of HLA-G by
JEG-3 cells [
37
], which could explain the increased cytotoxicity of NK-92 cells to JEG-3 cells
that we observed. Thus, the results obtained are consistent with the data in the literature.
TGF
β
, involved in the regulation of many physiological and pathological processes, is
one of the most powerful immunosuppressive cytokines [
42
]. The trophoblast JEG-3 cell
line produces TGF
β
[
43
,
44
]. Co E.C. et al. showed that decidual macrophages, by secreting
TGF
β
, can reduce the cytotoxicity of NK-92 cells and dNK cells towards K562 cells [
11
]. We
found that under the influence of TGF
β
, NK cells increased the expression of CD56 and
also reduced the expression of cytotoxic NKp30 receptors. TGF
β
is reported to suppress the
expression of the CD16 receptor by CD56bright NK cells, stimulating the differentiation of
CD56bright CD16-NK cells [
45
]. It has also been shown that TGF
β
inhibits the cytotoxicity
of NK cells against dendritic cells by reducing the expression of NKp30 by NK cells [
15
].
Thus, the changes in CD56 and NKp30 that we established can be regarded as the induction
of their regulatory phenotype.
At the same time, according to our data, TGF
β
causes a decrease in the number of
NK-92 cells with the NKG2A+ and KIR2DL4+ phenotype in monoculture. Hawke L.G. et al.
showed that after pNK cells were cultured with IL-15 and TGF
β
, they acquired an ILC1-like
phenotype and an altered expression of the transcription factors Eomes and T-bet [
46
]. In
response to TGF
β
in the culture system with IL-15, the expression of both activating and
inhibitory receptors, including NKG2A, by ILC1-like cells increased [
46
]. Thus, our data
complement the earlier results described in the literature.
In coculture with the JEG-3 cell line, TGF
β
also caused an increase in CD56 expression
by NK-92 cells, apposing their phenotype to the regulatory one. In the coculture system,
TGF
β
also affected trophoblast cells. It has been shown that TGF
β
can stimulate the
invasion of JEG-3 trophoblast cells [
47
]. The inhibition of signaling from TGF
β
in JEG-3 cells
leads to a decrease in the proliferation of trophoblast cells [
48
] and their ability to migrate
and invade [
43
,
49
]. Trophoblast JEG-3 cells express the immunoregulatory glycan-binding
protein galectin-1 (Gal1) [
50
]. Gal1’s ligand is T cell Ig and mucin domain-containing
protein 3 (Tim-3), expressed by dNK cells [
12
]. TGF
β
induces the upregulation of Tim-3
expression by dNK cells [
12
]. The Tim-3/Galecin-9 interaction inhibits the cytotoxicity of
NK cells against HTR-8 trophoblast cells [
12
]. However, we found that after the preliminary
cultivation of NK cells in the presence of TGF
β
for 24 h, the death of trophoblast cells did
not differ from that for the control.
In a study using mice, TGF
β
suppressed the NK cell-specific transcription factor Eomes
in spleen NK cells and induced them to acquire an ILC1-like phenotype [
51
]. Using ILCs
derived from the salivary glands of mice, it was demonstrated that ILC1-like cells contain
more granzyme B and C mRNA than NK cells. In knockout mice not expressing TGFβR2,
granzyme C expression was reduced in ILC1-like cells. [
51
]. In our work, we detected an
increase in the death of JEG-3 cells only after 96 h of the preliminary incubation of NK cells
with TGF
β
. It is possible that our results demonstrating the increased cytotoxicity of NK
cells after long-term exposure to TGF
β
are associated with the transdifferentiation of NK
cells into ILC1 cells with higher cytotoxicity.
In order to confirm the immunomodulatory effect of TGF
β
on NK cells in coculture
with trophoblast cells, we assessed the phenotype of pNK cells in our model system. Previ-
ously, we found that the cytotoxicity of pNK cells against JEG-3 trophoblast cells decreased
during the menstrual cycle, and did not differ in the secretory phase of the cycle from that

Int. J. Mol. Sci. 2022,23, 2387 12 of 18
in the first trimester of pregnancy [
44
]. In this regard, to assess the effect of trophoblast
cells on the pNK phenotype, we used PBMCs from healthy women, obtained in the second
phase of the menstrual cycle. We found that in the presence of the JEG-3 trophoblast cell
line, the expression of CD56 by pNK cells and the relative number of NKG2C+ pNK cells
were reduced. According to data in the literature, macrophages suppress the cytotoxic
function of NK cells through the secretion of TGF
β
[
11
,
52
,
53
]. PBMCs contain monocytes,
which, under cultivation, can differentiate into macrophages. Trophoblast cells can enhance
the secretion of cytokines by monocytes/macrophages. Awoyemi T. et al. showed that
syncytiotrophoblast microvesicles obtained from healthy pregnant women induced the
secretion of a spectrum of cytokines, including TNF
α
, IL-8, IL-6, VEGF, IL-1
β
and GM-CSF,
by monocyte-like cells of the THP-1 line [
54
]. The combined inhibitory effect of mono-
cytes/macrophages and trophoblast cells explains the observed decrease in the expression
of CD56 and the activation receptor NKG2C.
We also detected the reduced expression of CD56 and the activation receptor NKp44,
and a reduced number of pNK cells with the NKp44+ phenotype, when TGF
β
was added
to a PBMC monoculture as well as to a coculture of PBMCs with JEG-3 cells. However, in
our model with NK-92 cells, CD56 expression increased in the monoculture after cultivation
with TGF
β
. These differences may also be associated with the influence of monocytes on
the phenotype of NK cells in PBMCs. This assumption is consistent with the previously
established differences in the cytotoxicity of NK cells in PBMCs and NK cells isolated from
PBMC fractions [
55
]. It should be noted that a decrease in the expression of receptors by
pNK cells in the presence of TGF
β
affected not only the activation receptors but also an
inhibitory receptor—KIR2DL1. The observed decrease in KIR2DL1 expression may have
been associated with a general suppression of the expression of many NK cell receptors by
trophoblast cells in the presence of monocytes.
We found that in the presence of TGF
β
in the culture medium, pNK cells had a lower
expression of CD56 in coculture compared with monoculture. TGF
β
has been reported to
inhibit the production of progesterone and estradiol by JEG-3 cells [
56
]. It has also been
shown
in vitro
that as a result of exposure to pregnancy hormones, including progesterone,
estradiol and prolactin, THP-1 cells can change their secretory profile and begin to produce
cytokines characteristic of M2 macrophages [
57
]. It is possible that the inhibitory effect
of trophoblast cells and monocytes in the experiments with PBMCs was enhanced by
exogenous TGFβ.
4. Materials and Methods
4.1. Cell Cultures
We used the NK-92 cell line, which reproduces the main features of natural killer cells
(NK cells), in addition to the K562 myelogenous leukemia cell line and JEG-3 trophoblast
cells (ATCC, Gaithersburg, MD, USA); all cells were cultured in line with the manufacturer’s
recommendations. All the cell culture experiments were carried out in a humid atmosphere
at 37 ◦C under 5% CO2.
4.2. Peripheral Blood Mononuclear Cells
This study included 21 non-pregnant women with regular menses and an uncom-
plicated obstetric-gynecological and somatic history. The age range of the group was
29.1
±
6 years (M
±
SD). The group was divided into subgroups depending on obstetric his-
tories: healthy, non-pregnant women (subgroup 1, n= 8) and healthy, fertile, non-pregnant
women with a history of one or more pregnancies that ended in term delivery (subgroup
2, n= 13). Peripheral blood was obtained by venipuncture after an overnight fast, in
the secretory phase of the menstrual cycle, after the ultrasound monitoring of ovulation.
Blood sampling was carried out in the secretory phase, since we previously showed that
NK cells could change their functional activity in relation to trophoblast cells during the
menstrual cycle and in the secretory phase, and did not differ in cytotoxicity from the NK
cells of pregnant women in the first trimester [
58
]. The exclusion criteria for both subgroups

Int. J. Mol. Sci. 2022,23, 2387 13 of 18
were exacerbations of chronic diseases; the manifestation of acute inflammatory disease,
including antiphospholipid syndrome; external genital endometriosis stage 3–4; anomalies
in the development of the genital organs; obesity grade 2–3; a hereditary form of high-risk
thrombophilia; diabetes mellitus types 1 and 2; hormone therapy (particularly combined
oral contraceptives); or refusal to participate in the study program.
4.3. Cytokines
The cytokines IL-2 (‘Roncoleukine’, BioTech, Saint Petersburg, Russia), TNF
α
(50 IU/mL, R&D Systems, Minneapolis, MN, USA, cat. 210-TA), IFN
γ
(1000 IU/mL,
R&D Systems, Minneapolis, MN, USA, cat. 285-IF), TGF
β
(5 ng/mL, R&D Systems, Min-
neapolis, MN, USA, cat. 240-B) and IL-10 (10 IU/mL, R&D Systems, Minneapolis, MN,
USA, cat. 217-IL) were utilized as inducers.
4.4. Assessment of the Phenotype of the NK-92 Cell Line after Cultivation with JEG-3 Trophoblast
Cells and Cytokines
We used the method described previously in [
27
]. Before the experiment, JEG-3
cells were introduced into the wells of a flat-bottomed 24-well plate (200,000 cells/mL)
in complete DMEM growth medium (Biolot, St. Petersburg, Russia, cat.1.3.5.). After
24 h, the cells formed a confluent monolayer. Then, NK-92 cells were added to the wells
with JEG-3 cells (200,000 cells/mL). IL-2 (500 IU/mL) and one of the cytokines TNF
α
(50 IU/mL), IL-10 (10 IU/mL), IFN
γ
(1000 IU/mL) or TGF
β
(5 ng/mL) were added to the
wells. The cells were then cultured for 96 h, after which they were centrifuged (200
×
g,
22
◦
C, 10 min) and treated with an Fc receptor-blocking reagent (MACS, Teterow, Germany,
cat. 130–059-901). Next, the cells were treated with fluorescently-labeled monoclonal
antibodies against the phenotypic receptors CD45 (cat. 347464) and CD56 (cat. 557747);
cytokine receptors CD127 (cat. 560549), CD122 (cat. 557323) and CD215 (cat. FAB1471N);
differentiation receptors CD117 (cat. 333233), CD161 (cat. 556080), CD57 (cat. 333169)
and CD62L (cat. 559772); and cytotoxicity receptors CD94/NKG2A (cat. 555889), NKG2C
(cat. 748168), NKG2D (cat. 562498), NKp44 (cat. 558564), NKp30 (cat. 558407), KIR2DL3
(cat. FAB2014A), KIR2DL4 (cat. FAB2238P), KIR3DL1 (cat. FAB12251G), KIR2DL1 (cat.
FAB1844F) and KIR2DS4 (cat. 564375) (R&D Systems, Minneapolis, MN, USA; Becton
Dickinson, Franklin Lakes, NJ, USA). Isotype antibodies (R&D Systems, Minneapolis, MN,
USA; Becton Dickinson, Franklin Lakes, NJ, USA) were used as a control for nonspecific
binding. Receptor expression was assessed on a FACSCanto II flow cytometer (Becton
Dickinson, Franklin Lakes, NJ, USA).
4.5. Evaluation of the Cytotoxicity of the NK-92 Cell Line after Pre-Cultivation with Cytokines
NK-92 cells were pre-cultured in a 24-well plate in the presence of TNF
α
(50 IU/mL),
IL-10 (10 IU/mL), IFN
γ
(1000 IU/mL) or TGF
β
(5 ng/mL) for 24 h or 96 h. The incubation
with the cytokines was carried out for 24 h, since it was previously established that the
incubation of NK-92 cells with the supernatants of placenta samples led to a change in the
expression of surface cell receptors [
59
]. The incubation time of 96 h was chosen to match
the conditions of the experiments for the co-cultivation of NK-92 cells and JEG-3 cells. The
cells were then centrifuged (200
×
g, 22
◦
C, 10 min) and added to the target cells at a ratio
of 5:1 (effector:target), and the cytotoxic function of NK cells was assessed according to
the intensity of target cell death. JEG-3 trophoblast cells were used as targets, as described
previously [36].
4.6. Evaluation of the Phenotype of pNK Cells after Cultivation with JEG-3 Trophoblast Cells
and Cytokines
First, 24 h before the experiment, JEG-3 cells were placed in a 96-well flat-bottom plate,
with 20,000 cells per 100
µ
L of complete DMEM growth medium (BioloT, St. Petersburg,
Russia, cat.1.3.5.). One day later, peripheral blood mononuclear cells (PBMCs) were isolated
from peripheral blood using the standard method of centrifugation in a density gradient
solution of Ficoll (
ρ
= 1.077, BioloT, St. Petersburg, Russia, cat.1.2.8.1.). Then, the medium

Int. J. Mol. Sci. 2022,23, 2387 14 of 18
was removed from the wells with a trophoblast cell monolayer and PBMCs were placed
into the wells at 100,000 cells per 100
µ
L of complete growth medium (DMEM). To maintain
the viability of the PBMCs, we added IL-2 (200 IU/mL) to all the wells, while TGF
β
(5 ng/mL) was added to some of the wells, and the cells were incubated for 96 h. The plate
was then centrifuged for 5 min at 200
×
g, at 22
◦
C. The conditioned medium was removed
from the wells, and 100
µ
L of Versene solution (BioloT, St. Petersburg, Russia, cat. 1.2.3.2.)
was added. After 3 min, the cells were resuspended, transferred to a round-bottom plate,
and then centrifuged at 200
×
g, at 22
◦
C, for 5 min. The supernatant was removed, and a
reagent for blocking Fc receptors (MACS, Teterow, Germany, cat. 130–059-901) was added
to the cells. Then, the cells were treated with monoclonal antibodies against the CD3 (cat.
560176), CD56 (cat. 557747), CD45 (cat. 347464), CD14 (cat. 565283), KIR2DS4 (cat. 564375),
CD122 (cat. 557323), CD127 (cat. 560549), NKp44 (cat. 558564), NKG2D (cat. 562498)
(Becton Dickinson, Franklin Lakes, NJ, USA), KIR3DL1 (cat. FAB12251G), KIR2DL4 (cat.
FAB2238P), KIR2DL3 (cat. FAB2014A), KIR2DL1 (cat. FAB1844F), NKG2C (cat. FAB138P)
and CD215 (cat. FAB1471N) (R&D Systems, Minneapolis, MN, USA) receptors according
to the manufacturer’s instructions. PNK cells were analyzed in PBMCs as lymphocytes
with the CD45+ CD14-CD3- CD56+ phenotype using the previously published gating
strategy [
27
]. To assess cell death, some cells were treated with 7-amino-actinomycin
D (7-AAD) (cat. 420403, BioLegend, San Diego, CA, USA). The death of NK cells after
cultivation in mono- and coculture was no more than 2%. Isotype antibodies (Becton
Dickinson, Franklin Lakes, NJ, USA and R&D Systems, Minneapolis, MN, USA) were
used as controls. Receptor expression was assessed using a FACS Canto II flow cytometer
(Becton Dickinson, Franklin Lakes, NJ, USA).
4.7. Statistical Data Analysis
The data were statistically processed using the GraphPad Prism 8 program. We used
the Shapiro–Wilk test to assess the normality of the data distributions. Using the Bartlett
test, we estimated the homogeneity of variances. Due to the variances being unequal,
we further used non-parametric statistics for data analysis (Mann–Whitney U test and
Kruskal–Wallis test).
4.8. Ethical Approval
The research was conducted in line with the Code of Ethics of the World Medical
Association (Helsinki Declaration). The local ethical committee of the Research Institute of
Obstetrics, Gynecology, and Reproductology named after D.O. Ott approved the research
(protocol No. 107).
5. Conclusions
Here, we analyzed the effect of TNF
α
, IFN
γ
, TGF
β
and IL-10 on the receptor profile
and cytotoxic activity of NK cells. The pro-inflammatory cytokines IFN
γ
and TNF
α
stimulated the cytotoxic phenotype of NK cells. We showed that IFN
γ
promoted an
increase in the expression of the activation receptor KIR3DL1 by NK-92 cells. TNF
α
caused
a decrease in CD56 expression by NK-92 cells and stimulated their cytotoxic function.
The anti-inflammatory cytokine IL-10 did not affect the expression of receptors by NK-92
cells. In the case of co-cultivation with trophoblast cells, the effect of the cytokines on NK
cells was different. TNF
α
increased the expression of both the activating NKG2C receptor
and the inhibitory NKG2A receptor by NK-92 cells. In this regard, TNF
α
in combination
with contact interaction with trophoblast cells has a regulatory effect on NK cells and
contributes to the induction of a phenotype similar to that of memory cells. TGF
β
, in
the case of monoculture and coculture with trophoblast cells, stimulated NK-92 cells to
acquire a regulatory phenotype. When TGF
β
was added to a monoculture of PBMCs, as
well as to a coculture of PBMCs and JEG-3 cells, a reduced expression of CD56 and the
activation receptor NKp44 by pNK cells was revealed. Differences in the effect of TGF
β
in
the model using NK-92 cells and pNK cells may be associated with the possible influence of

Int. J. Mol. Sci. 2022,23, 2387 15 of 18
monocytes from the mononuclear fraction. Further studies are required to establish the full
effect of trophoblast cells on the expression of activating and inhibitory NK cell receptors
using primary dNK cells and/or decidual macrophages.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms23042387/s1.
Author Contributions:
Conceptualization, V.M. and D.S.; methodology, V.M., E.K. and Z.S.; investi-
gation, V.M., P.G., E.K., Z.S., A.D., K.M., V.Z. and E.T.; formal analysis, V.M. and D.S.; resources, S.S.
and I.K.; writing—original draft preparation, V.M.; writing—review and editing, V.M., D.S. and S.S.;
visualization, V.M., P.G. and K.M.; supervision, D.S. and S.S.; project administration, V.M. and D.S.;
funding acquisition, D.S. and S.S. All of the authors have reviewed and approved the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Russian Science Foundation Grant 21-15-00021 (influ-
ence of cytokines on NK-92 line cells after culturing with and without trophoblast cells), the Russian
Foundation for Basic Research Grant 20-015-00014 (cytotoxicity of NK-92 line cells after preculturing
with cytokines), the Ministry of Science and Higher Education of the Russian Federation, Research
Program 1021062512052-5-3.2.2 (clinical material acquisition, influence of cytokines on pNK cells
after culturing with and without trophoblast cells).
Institutional Review Board Statement:
This study was conducted in accordance with the Declaration
of Helsinki and approved by the Local Ethics Committee of the Federal State Budgetary Scientific
Institution Research Institute of Obstetrics, Gynecology, and Reproductology named after D.O. Ott
(Protocol No. 107, 15 March 2021).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in
the study.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
7-AAD—7-amino-actinomycin D; dNK—decidual NK cell; ILCs—innate lymphoid
cells; NK cell—natural killer cell; pNK—peripheral blood NK cell; PBMCs—peripheral
blood mononuclear cells.
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