The production of alpha/beta and gamma/delta double negative (DN) T-cells and their role in the maintenance of pregnancy

After exposure to hydrocortisone and dexamethasone, thymocytes become apoptotic and
undergo cell death 9], 10]. Whether or not sex steroids cause thymocyte loss by apoptosis was examined in a
number of studies in which animals were subjected to estrogen administration. Unfortunately,
the results were notable for their lack of consensus. Estrogen treatment in some studies
resulted in an increase in the rate of thymocyte apoptosis 11]–13], whereas in other reports, estrogen treatment produced little or no evidence of apoptotic
death 14], 15]. In a further study of the phenonomen, Zoller et al. 5] found that pregnant mice undergo extensive thymocyte loss and thymic involution without
thymocyte apoptosis ever taking place. In pregnant mice, the levels of estrogen range
between 7 ng/ml to 13 ng/ml 16]. Studies that reported a high incidence of thymocyte apoptosis injected the animals
with levels of estrogen far in excess of these values 11]–13]. Thus, without evidence to show that physiological levels of estrogen cause apoptosis,
this process can be ruled out as the reason for thymic involution and thymocyte loss.

Some investigators have proposed that thymocyte loss takes place because estrogen
blocks T-cell production at the precursor level. This premise came from a study in
which estrogen treatment resulted in an increase in the levels of the earliest CD44
⁺
progenitors and a depletion of all defined thymocyte subsets of CD4
⁺
and CD8
⁺
T-cells 17]. Other researchers have proposed that thymic involution is due to an estrogen-induced
reduction in early thymic progenitors 15]. These studies suggest the possibility that thymocyte loss is the result of an alteration
in T-cell production.

Martin et al. 18], 19], using light and electron microscopy, observed an estrogen-induced loss of thymocytes
in the subcapsular and deep cortex of the rat thymus. In the medullary region, they
found evidence of an increase in the vascular permeability of blood vessels located
near the corticomedullary junction. Lymphocytes were often seen migrating through
the enlarged walls of these blood vessels. They concluded that “the release of lymphocytes
from the thymus seems to be the main factor inducing thymic involution.” Others have
observed that the lymphatic vessels in involuted thymuses are packed with lymphocytes
(T-cells) 20], 21].

Although not identified as such, these lymphatic vessels would have to be efferent
lymphatic vessels, since the thymus lacks the afferent variety 22], 23], an important distinction.

Oner and Ozan 24] reported that prolonged treatment of female rats with either testosterone or estrogen
(daily for 3 weeks) caused extensive thymic involution. This involution was accompanied
by a loss of thymocytes in the subcapsular region as well as in the deep cortex. Blood
vessels in the thymic medulla were also enlarged, as was noted in the report by Martin
et al. 18], 19]. The most significant finding by Oner and Ozan, however, was the identification of
mast cells in connective tissue of the thymic capsule and in the stroma of the thymic
medulla. In untreated control rats, mast cells were sparsely distributed, whereas
in steroid-treated animals, they were increased in number and often found in clumps.
The fact that mast cells secrete vasodilators leaves little doubt as to the cause
of the increase in vascular permeability; which may be the reason why involuted thymuses
were packed with lymphocytes 20], 21]. As to the identity of these lymphocytes, studies of estrogen-injected 25], 26] and thymic-implanted nude mice 27] revealed that “thymocyte loss” was the result of the discharge of two subsets of
DN T-cells 25], 26]. One subset had a typical ?? T-cell receptor (TCR), and the other had a unique ??
TCR.

T-cell production

The thymus gland consists of two distinct lobes, each composed of a central medulla
and an outer cortex. Two layers of connective tissue, separated by a sinus, encapsulate
both lobes. In most species, the capsule gives rise to trabeculae that penetrate the
cortex and terminate at the corticomedullary junction, thereby providing a structural
link to the medulla. A basement membrane supports a specialized flattened epithelium
lining the subcapsule and trabeculae. Arteries travel within the capsule and then
either enter the cortex as arterioles or continue within the trabeculae until they
reach the corticomedullary junction, where they pass into the medulla. Arterioles
become progressively smaller and continue throughout the thymus as capillaries, undergoing
eventual transformation into venous capillaries and subsequent enlargement to form
postcapillary venules (PCVs). These venules ultimately lead to major blood vessels
that travel back to the trabeculae, where they leave in close proximity to the incoming
arteries 21], 22], 24], 28]–30].

The distribution of blood and lymphatic vessels (LVs) is not uniform. For example,
the cortex lacks PCVs, whereas the medulla contains a large number 21], 22]. In addition, the cortex contains a small contingent of branched LVs, located mainly
in the subcapsular region 31]. These vessels extend into the capsule and extralobular region and connect to efferent
lymphatic vessels (ELVs) 21], 22], 31]. In the medulla, LVs are more plentiful and are localized in the region of the corticomedullary
junction. These connect with ELVs in the trabeculae. Mast cells are absent from the
cortex, but are found nearby in the connective tissue of the capsule 24]. In the medulla, mast cells are located in proximity to both LVs and PCVs 24], 32], 33]. Notably, in the involuted thymus the number of mast cells is significantly increased
24], 34], 35].

T-cell progenitors produced in the bone marrow reach the thymus via the arterial branch
of the circulatory system. Upon entering the gland, they travel through arterioles
as well as arterial and venous capillaries until they arrive at the PCVs. The progenitors
then pass into the thymic stroma, using a process referred to as extravasation, or
diapedesis. Diapedesis takes place in vessels that have walls of endothelium and lack
a muscular layer 36], such as PCVs and LVs. Endothelial cells are unique in that lymphocytes are able
to insinuate themselves between cellular junctions, then pass either into, or out
of, the thymic stroma 37]–39]. Lymphocyte movement is aided by estrogen-activated mast cells through their production
of histamine and serotonin, which in turn, causes a widening of the cellular junctions
of the endothelial cells 36]. Diapedesis in PCVs is unidirectional and limited to lymphocyte movement from the
lumen into the thymus 40], 41]. For passage out of the thymus, the T-cells utilize LVs 42]–46], since these are capable of reverse diapedesis 47], 48].

Figures 1 and 2 are graphic representations of thymocyte development in pre- and postpubertal mice.
Shown in each figure are four spatially defined developmental stages in the cortex
that Lind and colleagues 49] have mapped using the progenitor markers, CD117 and CD25. Differential expression
of these two markers reflects developmental changes in the thymocytes as they move
outward from the corticomedullary junction into the cortex. In this process, thymocyte
movement is aided in large part by an interaction between chemokines produced by cortical
epithelial cells in specific areas of the cortex, and thymocyte chemokine receptors
50]. Stage 1 (CD117
⁺
CD25
^—
) begins at the corticomedullary junction and is characterized by thymocytes with
multilineage potential. These cells, in addition to giving rise to T lymphocytes,
can also evolve into B lymphocytes, as well as dendritic and NK cells. Cells that
reach stage 2 (CD117
⁺
CD25
⁺
) no longer have the ability to become B lymphocytes and NK cells, but can give rise
to ?? T-cells, ?? T-cells, and dendritic cells. Intracellular CD3? protein is detected
at this stage 51]. In addition, a significant amount of thymocyte proliferation occurs at stage 2.
Cells that reach stage 3 (CD117
^—
CD25
⁺
) are committed to T-cell lineage. Intracellular CD3? protein synthesis continues
unabated. TCR ? protein is first detected at this stage. Cells that express productive
rearrangements of TCR ? with an ? chain are selected to proliferate and proceed to
stage 4, a process termed as ? selection. At stage 4 (CD117
^—
CD25
^—
), thymocytes have reached the subcapsular region of the cortex with their TCR in
place and ? and ? binding components added to the CD3 complex. Most have traversed
the ?? TCR developmental pathway and are characterized as ?? CD4
^—
CD8
^—
double-negative (DN) T-cells. Thymocytes that have developed a ?? TCR are referred
to as ?? DN T-cells. Their numbers comprise 5-10 % of total DN T-cells 27], 52].

Fig. 1. Proposed pathways for the production of T-cells in prepubertal mice. Progenitor cells
enter the thymus via postcapillary venules (PCVs) located in the medulla and as T-cells
leave by way of efferent lymphatic vessels (ELVs) located in the subcapsular cortex
and in the medulla. In prepubertal mice, the majority of thymocytes traverse the classic
developmental pathway and as SP T-cells enter the lymphatic system (LS) (solid black arrows) via ELVs located in the medulla. Lesser numbers of thymocytes enter the LS (dashed black arrows) as DN T-cells through ELVs located in the subscapular region

Fig. 2. Proposed pathways for the production of T-cells in postpubertal mice. Progenitor cells
enter the thymus via postcapillary venules (PCVs) located in the medulla and as T-cells
leave by way of efferent lymphatic vessels (ELVs) located in the subcapsular cortex
and in the medulla. In postpubertal mice, mast cell activation (red dots) results in large numbers of thymocytes exiting the classic pathway as DN T-cells
and entering the LS (solid red arrows) via ELVs located in the subcapsular region. Lesser numbers of thymocytes remain
in the classic pathway and enter the LS (dashed red arrows) as SP T-cells via ELVs located in the medulla

DN T-cell pathway

Gamma/delta T-cells are not found in the thymus beyond stage 4 of development 51]. This suggests: 1) an absence of thymic tissue specifically dedicated to the continuation
of their chemokine-facilitated travel; and 2) a strong probability that they leave
the thymus directly after they are produced. Lymphatic vessels located nearby in the
subcapsular cortex are very likely their means of exit. In mice, the DN pathway is
operational shortly shortly after birth, with DN T-cells being found in the liver
and spleen of 4-day-old animals 52], 53]. Notably, the levels of ?? DN T-cells exceed that of ?? DN T-cells by a factor of
4:1. Shown in Fig. 1 are the proposed exit pathways of ?? DN T-cells and ?? DN T-cells in prepubertal
mice. As is indicated, most T-cells leave the thymus via ELVs located in the medulla
(solid black arrows). However, in postpubertal mice (Fig. 2) a large number of ?? DN T-cells and ?? DN T-cells exit the thymus via ELVs located
in the subcapsular cortex (solid red arrows) as the result of a sex steroid-induced
activation of thymic mast cells.

Estrogen activation of mast cells takes place via a membrane-associated (non-genomic)
estrogen receptor-? (ER-?) 7]. This activation results in an influx of extra-cellular calcium and the synthesis
and release of granules of histamine and serotonin 8]. Mast cell activation can be achieved with concentrations of estrogen between 10
^?11
 M and 10
^?9
 M (2.7 pg/ml to 270 pg/ml) 54]. Testosterone activation requires levels that are 10 times that of estrogen 55]. Activation by the weak androgen, dehydroepiandrosterone (DHEA), necessitates levels
that are 1000 times that of estrogen 56], 57]. Dihydrotestosterone (DHT) is also a mast cell activator 58]. Progesterone is an inhibitor of estrogen activation 59].

In postpubertal animals, endogenous sex steroids attain levels that are fully capable
of activating thymic mast cells. For example, circulating levels of testosterone in
male mice and rats average 18.7 ng/ml and 5.8 ng/ml, respectively 60]. In nonpregnant female mice and rats, the levels of estrogen are 66 pg/ml and 30.6 pg/ml,
respectively 61], 62], and in pregnant mice, estrogen levels range from 7 ng/ml to 13 ng/ml 16]. Strong evidence that the ER-? plays a role in estrogen-induced thymic involution
is indicated by studies of estrogen receptor knockout mice (ERKO). In these animals,
the ER-? is nonfunctional; consequently, the thymus undergoes only minimal estrogen-induced
involution 63], 64].

Classic T-cell pathway

In contrast to the fate of ?? DN T-cells, ?? DN T cells retain the option of continuing
their development in the thymus. This choice is exercised when CD4 and CD8 markers
are expressed, and ?? DN T-cells become CD4
⁺
CD8
⁺
double-positive (DP) T-cells. In utilizing this option, DP T-cells apparently lose
the ability to access the DN pathway. This is either because they are restricted from
doing so or have left the area of the subcapsular LVs. Abo’s group reports a total
absence of DP T-cells in the pool of SP T-cells and DN T-cells found in the liver
of estrogen-injected mice 25]. In the next developmental stage, DP T-cells undergo positive selection, a procedure
concurrent with the production of two subsets of single-positive (SP) MHC restricted
T-cells. These subsets are CD4
⁺
(class II MHC-restricted) and CD8
⁺
(class I-MHC restricted) T-cells, and as such, they continue on into the medulla.
Here they undergo negative deletion, a process in which their ?? TCRs are exposed
to ectopic self-antigens. Production of these antigens is under the direction of the
autoimmune regulator (Aire) promotor 65]. Fully mature CD4
⁺_Helper
, CD4
⁺
CD25
⁺
Foxp3
⁺_Regulatory
, and CD8
⁺_Cytotoxic
T-cells exit the thymus via LVs located in the medulla (Fig. 1, solid black arrows; Fig. 2, dashed red arrows).

Interaction between DN and SP pathways

It should be noted that the permeability of all LVs and PCVs is increased through
the combined action of sex steroids and mast cells. This results in an increased entry
of T-cell progenitors and an enhancement in the exit rate of DN T-cells. To gain an
appreciation of the levels of thymocytes that exit the thymus via the DN pathway,
one only has to measure the total number of thymocytes prior to, and after castration.
Fortunately, this has been done by a number of researchers. For example, Pesic et
al. 66] reported that thymocyte levels in castrate and intact 60-day-old Albino-Oxford male
rats were 1050 × 10
⁶
and 650 × 10
⁶
, respectively. This would suggest that mast cell activation has facilitated the exit
of 38 % of total thymocytes. Notably, these thymocytes were reported to originate
from the cortex. In a study of intact and castrated 60-day-old female Sprague–Dawley
rats 57], the results indicated that estrogen caused 44 % of total thymocytes to exit via
the DN pathway. Findings from a third study of male and female adult Wistar-albino
rats 24] revealed that testosterone and estrogen affected a reduction of 31 % and 30 % of
total thymocytes, respectively. These studies demonstrate the effect of sex steroids
in altering the dynamics of T-cell production. In the castrate animal, the thymus
produces mainly SP T-cells. Their production time takes 3–5 days in the cortex and
12–16 days in the medulla 67], for a total of ~21 days. In the intact animal, a significant number of DN T-cells
exit the thymus via the DN pathway. Their total production time is 3–5 days. In these
animals, the reports of a reduction in thymocyte levels of?~?35 % 24], 57], 66] strongly indicates that progenitor replacement does not keep pace with DN T-cell
production.

Pesic et al. 66] also measured thymocyte levels in the cortex and medulla of intact and castrate male
rats. With this information we were able to examine the effect of the discharge of
DN T-cells in altering the levels of SP T-cells. For example, in the castrate animal
(Fig. 3) a comparison between thymocyte levels in the cortex and medulla indicates that 2 %
of total thymocytes leave via the DN pathway, and 11 % reach the medulla to become
SP T-cells. Without castration (Fig. 4), a similar comparison suggests that 38 % of total thymocytes exit via the DN pathway
and only 7 % reach the medulla. Thus, the production of DN T-cells is the result of
a proverbial “fork in the road” of T-cell development. Thymocytes can either leave
the thymus as DN T-cells, or they can remain in the classic T-cell pathway and exit
as SP T-cells. Their pathway of development is determined by sex steroids. For example,
during pregnancy when estrogen levels are at their highest, large numbers of T-cells
utilize the DN pathway. As a consequence, the production of SP T-cells is at its nadir
15]. We estimate that during pregnancy only 2 % of total thymocytes reach the medulla.

Fig. 3. Production of DN T-cells and SP T-cells by castrate adult animals. Shown are the percentages
of DN T-cells and SP T-cells produced by castrate adult animals. The numerical values
were determined from the data of Pesic et al. 66]

Fig. 4. Production of DN T-cells and SP T-cells by intact adult animals. Shown are the percentages
of DN T-cells and SP T-cells produced by intact adult animals. The numerical values
were determined from the data of Pesic et al. 66]

DN T-cells

DN T-cells do not undergo positive selection (Figs. 1 and 2). Consequently, they lack MHC restriction. This factor, in combination with their
unique TCR, produces binding characteristics for ?? DN T-cells that differ substantially
from that of MHC restricted ?? T-cells. Where the latter bind to fragments (epitopes)
of foreign antigen held within the cleft of a class I or class II MHC molecule 68], ?? DN T-cells do not. Instead, their binding to foreign antigen is based on the
conformational shape of the intact antigen, similar to that of antibodies, and independent
of MHC involvement 69].

There are three major subsets of ?? DN T-cells, one of which is cytolytic. In humans
this subset has been characterized via its TCR as a V?9V?2 T-cell 69], 70]. When activated, they secrete interleukin-2 (IL-2), interferon-? (IFN-?), and tumor
necrosis factor-? (TNF-?) 71]. These cytokines promote inflammation, cytotoxicity, and delayed-type hypersensitivity
(DTH) 72]. V?9V?2 T-cells are unconventional in that non-proteins such as isoprenoids and alkylamines
cause their activation 69]. Their venue of immunological activity is in the peripheral bloodstream 70], 71]. Here they have an important role in both tumor cell surveillance and anti-infective
immunity 73]. The second subset of ?? DN T-cells has all the characteristics of the V?9V?2 T-cells,
except they are not cytolytic. The reason they are not is because they have an intermediate
and incompletely expressed TCR/CD3 binding complex 74]–76]. Henceforth they will be referred to as ?? DN (int TCR/CD3) T-cells. Rather than
being in the bloodstream, these T-cells reside in the intraepithelial lymphocyte compartments
of specific tissues such as skin, intestine, respiratory tract and uterus 69], 74]. The third subset of ?? DN T-cells are regulatory. In mice they are characterized
as V?6V?1 regulatory T-cells 77]. Activation of these ?? DN regulatory T-cells results in the production of IL-10
and transforming growth factor-? (TGF-?) 76], 78]. These cytokines control the action of cytotoxic T cells, NK cells, macrophages,
dendritic cells and B cells 79]. The ?? DN regulatory T-cells are also restricted to the intraepithelial lymphocyte
compartments of specific tissues 79]. In the uterus they play a significant role in the maintenance of pregnancy.

Alpha/beta DN T-cells are cytolytic 80] and produce IL-4, IFN-?, and TNF-?, but not IL-2 81]. These T-cells have a significant role in the control of intracellular bacterial
infection 82].

Immunomodulation, DN T-cells, and the maintenance of pregnancy

The maintenance of pregnancy depends, to a large extent, on the avoidance of maternal
rejection. This is dealt with through the construction of an immunological barrier
using cells that lack the ability to express classical HLA-A and HLA-B products 83]. This produces a protective cocoon (trophoblast) in which MHC class I and MHC class
II molecules are either missing or non-functional 84]; as a consequence, the processing and presentation of antigens by MHC molecules cannot
take place. SP T-cells are thus eliminated as a rejection factor, leaving only ??
DN T-cells to respond to the trophoblast. Instead of rejection, however, these T-cells
are essential for the maintenance of pregnancy. The complexity of their overall role
and the need for coordination requires extensive communication between ?? DN T-cells
and the decidua and trophoblast. The trophoblast, for example, initiates contact with
a variety of immune cells through its production and release of chemokines. These
are small proteins that act as ligands to immune cell receptors. The binding of these
unique ligands to specific receptors results in the production of adhesion molecules
by respondent cells, thereby giving them the means to adhere to the endothelium of
blood vessels. With this ability they are able to follow a chemokine concentration
gradient to its source 85]. Cytokines produced by ?? DN T-cells, in contrast, encompass a broader application
than chemokines in that they influence the growth and receptivity of specific cell
populations.

The trophoblast attracts immune cells to the fetal-maternal interface through its
production of the chemokines CXCL12 and CXCL16. For example, CXCL12 recruits NK cells
that have CXCR3 and CXCR4 receptors 86], 87], and CXCL16 recruits ?? T-cells, ?? DN T-cells, and monocytes through its interaction
with CXCR6 receptors 88]. Analyses of the decidua during early to mid-pregnancy has identified the presence
of the following cells: 1) ?? DN regulatory T-cells; 2) ?? DN (int TCR/CD3) T-cells;
3) CD8
⁺
cytotoxic T-cells; 4) CD4
⁺
CD25
⁺
Foxp3
⁺
regulatory T-cells; 5) NK cells; 6) dendritic cells; 7) macrophages; and 8) neutrophils
75], 89], 90]. These cells have all reached the decidua via the cardiovascular system, with two
exceptions. The exceptions are ?? DN regulatory T-cells and ?? DN (int TCR/CD3) T-cells.
These two subsets are part of a group that obtains access to their target tissues
via the lymphatic system 69], 74], 75], 78], 91].

In nonpregnant women, mice, rats, and rabbits, the lymphatic system does not extend
beyond the myometrium 92]–94]. Therefore, during early pregnancy, ?? DN regulatory T-cells and ?? DN (int TCR/CD3)
T-cells are unable to respond to CXCL16 until lymphangiogenesis (lymphatic vessel
growth) has linked the endometrium to the lymphatic system. As a consequence, these
T-cells are the last to reach the fetal-maternal interface. Their late arrival indicates
the likelihood that lymphangiogenesis does not require their input, at least at this
point. Their participation in the process comes later and is essential for the maintenance
of pregnancy.

Gamma/delta DN cytolytic T-cells are found in the uterus during the early stages of
pregnancy 95]. Their presence in this location is very likely due to CXCL16. However, the main
function of these T-cells is to detect and destroy bacteria, and they are highly cytolytic.
Thus, it is unusual for these cells to be in close proximity to the trophoblast without
causing its destruction 96]. To protect the trophoblast could be the reason why a large number of CD4
⁺
CD25
⁺
Foxp3
⁺
regulatory T-cells reside in the decidua 89]. These regulatory T-cells are fully capable of eliminating ?? DN cytolytic T-cells
97], 98]. It is noteworthy that Foxp3
⁺
regulatory T-cells are among the first immune cells to enter the uterus, indicating
that they are in place prior to the entry of the ?? DN cytolytic T-cells 99]. Levels of Foxp3
⁺
regulatory T-cells undergo a significant increase during pregnancy 5], 89], 100], 101], with the decidua being the major recipient of their enhanced production 89]. It should be noted that this form of protection for the trophoblast has an upper
limit since excess numbers of peripheral ?? DN cytolytic T-cells can cause abortion
96], 102], 103]. It was not reported in these studies if the increase in ?? DN cytolytic T-cells
was due to acute bacterial infection 104], 105]. Putative evidence of the involvement of Foxp3
⁺
regulatory T-cells in preventing abortion is indicated by reports that women with
decreased levels of these T-cells suffer from recurrent miscarriages 106]–108].

NK cells play a significant role in the creation of blood and lymphatic vessels. Their
major responsibility is to produce a large number of cytokines. These include vascular
endothelial growth factor (VEGF), fibroblast growth factor (FGF), TNF-?, IFN-?, and
the angiopoietins, to name a few 109]. Blood-borne NK cells are cytolytic and fully capable of destroying the trophoblast
110]. However, unlike the ?? DN cytolytic T-cells, they are not eliminated. Instead, they
are converted into noncytolytic NK cells. This transformation is under the control
of TGF-?, and involves converting cytolytic CD56
^dim
CD16
⁺
peripheral NK cells (CD16
⁺
pNK cells) into noncytolytic CD56
^bright
CD16
^—
uterine NK cells (CD16
^—
uNK cells) 111]–113]. The initial source of TGF-? for pNK cell conversion is provided by the male, and
TGF-? reaches the decidual area via the ejaculate 114]–116]. TGF-? is also produced by decidual stromal cells 112]. However, the overall supply of TGF-? is not inexhaustible. The TGF-? derived from
the ejaculate is limited for obvious reasons, and the ability of stromal cells to
produce the cytokine is seriously compromised. This is because TGF-? is involved in
two simultaneous and conflicting operations. In addition to converting pNK cells into
uNK cells, TGF-? is also involved in implantation. Its role in this process is to
initiate the apoptotic destruction of decidual stromal cells.

Shooner et al. 117] noted that stromal cells of the pregnant rat uterus undergo a TGF-?-induced increase
in apoptosis between day 5 and day 14 of pregnancy. During this period, the loss in
stromal cells is correlated with decreased production of the two isoforms, TGF-?1
and TGF-?2. After day 14, only limited quantities of TGF-? are produced by stromal
cell survivors. Without replenishment, the decrease in TGF-? could have a serious
impact on the transformation of pNK cells to uNK cells. Red-Horse 94] noted that lymphatic vessels in the endometrial area of pregnant mice begin their
development between embryonic day 9.0 and day 9.5. This would indicate that these
lymphatic vessels have?~?5 days to complete their development before TGF-? is seriously
depleted. This timeframe is critical since ?? DN regulatory T-cells, a major source
of TGF-?, can only reach the fetal-maternal interface via the newly-formed lymphatic
vessels.

TGF-? is regarded as a pleiotropic cytokine. This characteristic is obvious during
the maintenance of pregnancy. Here, the cytokine has a significant impact on lymphangiogenesis
by controlling levels of pNK cells 112]. However, while TGF-? is performing this function it is undergoing self-destruction
by initiating the apoptosis of decidual stromal cells 117]. Both processes are essential for the maintenance of pregnancy. The prospect of the
cytokine being depleted during implantation is troublesome. One could visualize scenarios
in which the levels of stromal cells were lower than normal, or where TGF-?-induced
stromal cell apoptosis occurred at a faster rate. In these instances, implantation
would be successful, whereas a scarcity of TGF-? could alter the formation of lymphatic
vessels. If this occurred, it would prevent ?? DN regulatory T-cells from reaching
the fetal-maternal interface. The loss of a major source of TGF-? could impede the
conversion of pNK cells to uNK cells. Notably, a number of studies have reported that
excess levels of pNK in pregnant women are highly correlated with recurrent spontaneous
abortion 118]–125].