Regulation of the innate immune cells during pregnancy: An immune checkpoint perspective

Abstract The foetus can be regarded as a half‐allograft implanted into the maternal body. In a successful pregnancy, the mother does not reject the foetus because of the immune tolerance mechanism at the maternal‐foetal interface. The innate immune cells are a large part of the decidual leukocytes contributing significantly to a successful pregnancy. Although the contributions have been recognized, their role in human pregnancy has not been completely elucidated. Additionally, the accumulated evidence demonstrates that the immune checkpoint molecules expressed on the immune cells are co‐inhibitory receptors regulating their activation and biological function. Therefore, it is critical to understand the immune microenvironment and explore the function of the innate immune cells during pregnancy. This review summarizes the classic immune checkpoints such as PD‐1, CTLA‐4 and some novel molecules recently identified, including TIM‐3, CD200, TIGIT and the Siglecs family on the decidual and peripheral innate immune cells during pregnancy. Furthermore, it emphasizes the role of the immune checkpoint molecules in pregnancy‐associated complications and reproductive immunotherapy.


| INTRODUC TI ON
The crosswalk between the mother and foetus during pregnancy using immunological markers remains enigmatic. The foetus has been first described as an allograft developing in an immunologically competent maternal host in 1953 by Sir Peter Brian Medawar. 1 Various immunomodulatory cells are recruited into the placenta or proliferate locally, and residing in the decidua, as well as the cell surface receptors and secreted molecules, and are involved in the induction and maintenance of the tolerance throughout pregnancy.
The innate immune cells, such as the natural killer cells (NK cells), dendritic cells (DCs) and macrophages, are abundant at the early maternal-foetal interface, where they differentiate into tolerogenic subsets with different functions. 2,3 The trophoblast cells selectively express a set of unique MHC molecules (HLA-C, HLA-E, HLA-G), which allow simultaneous "loss-of-self" recognition and refusal to escape from the NK cells and macrophages through ligand-binding, like the KIR family, NKG2 family, and LILR family. [4][5][6] Immune checkpoint molecules are referred to as a series of coinhibitory receptors that are expressed on the immune cells regulating the degree of immune activation playing a positive role in inducing transplantation tolerance, tumour immune escape and autoimmune prevention. [7][8][9] Some immune checkpoint molecules are observed on the innate immune cells during pregnancy playing a fundamental role in a successful pregnancy. However, the underlying mechanisms remain elusive. In this review, we investigated and summarized the current knowledge on the role of maternal-foetal immunotolerance based on innate immune checkpoint molecules.

| INNATE IMMUNE CELL S AT THE MATERNAL-FOE TAL INTERFACE
Immune cells are important components of the decidual microenvironment. A large part of the decidua comprises the maternal leukocytes (approximately 40%). 2,3 In the first trimester of pregnancy, the innate lymphoid cells (ILCs) with decidual NK cells (dNKs) comprise up to 70% of the decidual leukocytes, followed by the decidual macrophages (dMs) (20 to 25%) and T cells (3%-10%), 10 indicating that the innate immune cells play an indispensable role in the maternalfoetal interface.

| Innate lymphoid cells
Five main ILC subsets have been identified in the decidua, including the dNK1-3, group 3 innate lymphoid cells (ILC3s) and proliferating NK cells. 11 Unlike the peripheral blood NK cells and other tissue-resident NK cells, the dNKs are have been typically defined as the CD56 superbright cells. 12,13 dNK1 was the most abundant subset identified in the cryopreserved ILCs (30%), followed by dNK2 (15%) and dNK3 (15%). dNK1 has larger granules, including perforin and granzyme, with higher expression of KIR, while dNK2 and dNK3 produce more cytokines. 11 However, the origin of dNKs remains unclear. There are different pieces of evidence suggesting that dNKs are not only recruited by the CD56 bright CD16 − NK cells or by differentiation of CD56 dim CD16 + NK cells in the peripheral blood but also from the immature NK precursors or mature hematopoietic CD34 + precursors in the uterus. [14][15][16] DNKs can control the extravillous trophoblast invasion independent of a non-cytotoxic mechanism and promote vascular remodelling. NK cells interact with and modulate other maternal immune cells. DCs can be primed by the activated NK cells, which inducing a protective CD8 + T-cell response. 3 The ILC3s are the RAR-related orphan receptor gamma t + (RORγt + ) and secrete IL-22, IL-17A, IL-8 and TNFα. 17 In humans, two subsets of ILC3s have been classified as natural cytotoxic receptor-negative (NCR − ) and NCR ILC3s (also referred to as lymphoid tissue inducer (LTi)-like cells). The NCR + ILC3s population is dominant in the human uterus and is involved in the recruitment and the survival of decidual neutrophils. 18,19

| Gamma delta T cells
A resident T-cell population in the decidua is significantly enriched in the gamma delta T cells (γδTs) compared to the blood. 22 The γδTs have been implicated in the responses to infectious diseases, in the regulation of immune responses, and in tissue homeostasis and repair. 23 The percentage of γδT in the decidual CD3 + T cells is controversial. 24 The decidual γδTs are large granular lymphocytes enriched in the cytoplasmic granules that do not express the CD4 or CD8 markers. The TCR of the decidual γδTs is composed of a V γ 9 / V δ 1 chain, which is different from the peripheral blood lymphocytes expressing the V γ 9 / V δ 2 chain. 25 Early pregnancy can also induce the activated and terminally differentiated proinflammatory γδ T-cell effectors to recruit at the maternal-foetal interface with a variety of TCR components. 26 These data confirm that the decidual γδT can suppress the population of the other cells and promote the proliferation and invasion of human trophoblast cells via IL-10 and TGFβ. 24,27

| Mucosal associated invariant T cells
The mucosal-associated invariant T (MAIT) cells are a type of immune cell subset that is relatively rich in intervillous cells in the decidua and term pregnancy compared to the peripheral blood. The MAIT cells are anti-microbial and tissue repair T cells, and cannot respond to the same HLA molecules, representing the ideal characteristics of the effector cells in the foetal mother interface. 28 Decidual MAIT cells produced higher levels of granzyme B and similar levels of the IFNγ-responsive Escherichia coli compared to the peripheral MAIT cells. 29 The intervillous MAIT cells can interact with the foetal macrophages located in the injured regions of the villous tissue and promoting tissue repair. 30

| IMMUNE CHECKP OINT MOLECULE S
The immune checkpoints are inhibitory pathways in the immune system and are regulated by the ligand/receptor interactions. They play an important role in maintaining the autoimmune tolerance and regulating the duration and amplitude of the physiological immune response to avoid damage and destruction of the normal tissues caused by the immune system. There are few reviews that focus on the area of immune checkpoints. 31,32 Although the immune checkpoint molecules were originally been identified in the T cells, the innate immune cells are becoming a newly emerging target. The connection. [38][39][40][41][42][43] Tim-3 is a member of the Tim family and was first identified as a receptor expressed on the CD4 + and CD8 + producing IFNγ. 44 Tim-3 consists of five atypical cysteine amino-terminal immunoglobulin variable domains, a mucin stem, a transmembrane domain, and a cytoplasmic tail. 45 The Tim-3 cytoplasmic tail has no classical inhibitory signal transduction motifs, such as ITIM or ITSM. 46 The expression of Tim-3 is not limited to the T cells, but different types of immune cells, including the B cells, Tregs, NK cells, DCs, monocytes and macrophages are also expressed. 47 Four ligands have been reported to bind to Tim-3, including galectin-9 (Gal-9), phosphatidylserine, CEACAM1 and high mobility group protein B1 (HMGB1). 47 The Tim-3-galectin-9 interaction plays a role in inhibiting immune response.
The lymphocyte activation Gene-3 (LAG-3) is a cell surface molecular protein encoded by the LAG-3 gene, which is mainly expressed in activated T cells, NK cells and plasma cell-like dendritic cells. The main ligand of LAG-3, MHCII, which negatively regulates the proliferation and activation of the T cells, is similar to that of CTLA-4 and PD-1. Fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand-independent from MHC-II. 48 It plays a role in inhibiting the function of the Treg cells and maintains the tolerance of the CD8 + T cells. LAG-3 in the Treg cells is essential for the T-cell exhaustion. 49  but it has a 67aa intracellular segment containing three tyrosine residues. 58 After activation by CD200R, the third tyrosine will be phosphorylated, leading to the aggregation and phosphorylation of Dok-1 and Dok-2 with subsequent combination of RasGAP and Ship.
In macrophages and rod cells, this reaction can inhibit the phosphorylation of ERK, JNK and p38. The mouse experiments have shown that the activation of CD200R in macrophages could inhibit the autoimmune response. These results suggest that the CD200-CD200R signal is involved in the inhibition of the myeloid cell function. [58][59][60][61][62] TIGIT was first identified as an immune checkpoint rheostat that suppresses the activation of the T cells using a bioinformatics algorithm. 63 TIGIT was named differently, including WUCAM, and later changed to Vstm3 and then to TIGIT. 64

| The functions of the immune checkpoint molecules in normal pregnancy
The functions of the immune checkpoint molecules in innate immune cells during pregnancy are summarized in Table 1. During pregnancy, PD-1 expression has been identified in a broad spectrum of decidual innate cell subsets including macrophages, NK cells,

NKT-like cells, ILC3, γδT cells and MAIT cells. DM polarization and
function depend on the PD-1 signalling during early pregnancy. 69 The polarization of GM-CSF-differentiated macrophages towards the M2 phenotype can be promoted by PD-1 agonist (PD-L1 Fc), an activator of the PD-1, which has higher phagocytic activity and can be reversed by the PD-1 blockade via reprogramming metabolism, consequently decrease inflammation. 70 The soluble PD-L1 has been shown to increase during pregnancy and may play a role in immune tolerance, which is critical for the foetus, which has already been confirmed. 71,72 Compared to the peripheral cells, the dNKs, dNKTs and decidual γ/δ T cells showed upregulated PD-1 expression and reduced cytotoxic potential, 73 especially the CD56 + dNKs. 74 The first evidence that human decidual ILC3s express a functional PD-1 has been confirmed. Both CD56 + ILC3s and LTi-like cell subsets are sup-  expressing high levels of PD-L1, suggesting that PD-1/PD-L1 interaction may regulate ILC3 function at the foetal-maternal interface.
Notably, ILC3s decrease the PD-1 expression significantly with the development of pregnancy. The results showed that the expression of PD-1 on ILC3s (especially LTi-like cells) is indirectly related to the pregnancy stage. 75 The MAIT cells could play an important role in protecting the foetus from bacterial infections and maintaining homeostasis at the foetal-maternal interface. 28 The decidual MAIT activation rather than M1 polarization during early pregnancy. 98,99 In the murine pregnancy, Tim-3 + pNKs exhibit immunosuppressive functions, including increasing the production of anti-inflammatory cytokines, decreasing the production of proinflammatory cytokines and impairing cytotoxic activity. 100 The Tim-3 + peripheral NK cells Sialic acid (Sia) is dispensable for protecting the foetus from maternal complement attack. 114 Moreover, Sia can combine with Siglecs as a ligand, which is critical or restraining the immune response. 50 The ability of the trophoblast invasiveness is suppressed by the interaction between the Siglec-6 and Glycodelin-A protein (GdA), a glycoprotein that is abundantly expressed in the decidua and participates in defence and placental development, which downregulating the ERK/c-Jun signalling pathway. 115 Siglec-7 mediates the binding of the GdA in a sialic acid-dependent manner to induce the polarization of the monocytes into dMs to regulate immune tolerance and placental development. 116 Co-localization of Siglec-10 with CD24 observed at the maternal-foetal interface especially in the endometrial glands and decidual cells close to extracellular trophoblasts suggests a possible role in maintaining immune tolerance. 117 In murine pregnancy, the expression of CD200 is expressed in the decidual cell subsets and trophoblasts of the mouse uterus. 118 Embryo resorption is inhibited by CD200 expression in the trophoblasts. 118 Macrophages and mast cells also express CD200R.
Upregulation of CD200 alters DCs to promote Treg cell development and suppress the macrophages and mast cells. 119 Mesenchymal stem cells (MSCs) also express CD200 and the interaction with CD200R on the decidual M1 and CD200 on MSCs promoted the shift of the proinflammatory macrophage phenotype to an anti-inflammatory phenotype. 120 The limited expression of CD200R subtypes at the maternal-foetal interface suggests that the interaction of CD200 and CD200R may play an important role in determining the successful outcome of pregnancy. 121 In human pregnancy, compared to the non-pregnant women, the population of peripheral blood monocytes in early pregnant women is increased while the total number of leukocytes and lymphocytes show no significant changes. These monocytes are found to be upregulated the expression of CD200 and CD200R. 122 CD200R can regulate the cytotoxic activity of the NK cells during human pregnancy. 123 The expression of CD200 and CD200R on the CD1c + myeloid and BDCA-2 + lymphoid DCs in the first trimester of normal pregnancy was significantly higher than that in the non-pregnant women, suggesting that the myeloid and lymphoid DCs increased tolerogenic properties in early pregnancy. 124 After 5 weeks of gestation, the CD200 tolerance signal molecule and its receptor CD200R1 were expressed in the human placental villous trophoblasts and implant decidua. 125 In the peripheral blood, TIGIT is expressed on the γδT cells and Tim-3-Gal-9 signalling-mediated immunoregulation by pNKs plays a critical role in maternal-foetal immune tolerance and suggests that Tim-3 abundance on pNKs is a potential biomarker for RSA diagnosis. 101 Clinical data show that abnormal Tim-3 levels on pNKs might be associated with RSA 103 and interference of cytokine profiles and increased cytotoxicity are observed in the Tim-3 + dNKs. 102 In human miscarriages and murine abortion-prone models, dNKs decrease the percentage of Tim-3-expression and the production of the Th2-type cytokines while Th1-type cytokines are increased. 102 DSCs decrease the expression of Tim-3 in the miscarried women who miscarried. Tim-3 is a key regulator of DSCs and s shows a potential role as a target for the treatment of spontaneous abortion. 133 Proinflammatory cytokine production and cell apoptosis in DSCs are induced by the activation of TLR signalling, which could be inhibited by Tim-3 in the ERK1/2 pathway-dependent manner and suppressing of NF-κB activation. 133 The CTLA-4 gene is first involved in maintaining immune tolerance at the maternal-foetal interface and plays a role in inducing RSA. 134 The surface density of CTLA-4 on decidual CD26 + lymphocytes was lower in the model of stress-induced abortion and returned to normal levels by using inhibiting of DPP IV. 135 The proportion of decidual CD4 + CD25 + T cells was significantly lower. 109 The CD4 + CD25 + Treg cells increase the induction of IFN-y to upregulate the IDO expression in DCs and monocytes in normal pregnancy, but downregulate in RSA. 136 CD200 and its receptor CD200R1 decrease at 7-9 weeks of gestation in RSA. 125  in invasive cytotrophoblasts, which is unique in spontaneous PE. 151 The DCs decreased the CD200R expression. 124,152 There is a higher risk of PE to inhibit PD-L1 and CD200, which suppresses the IDO expression in the placenta. 153 The peripheral MAIT cells expressing

| IMMUNE CHECKP OINTS IN REPRODUC TIVE IMMUNOTHER APY
Abortion is the most common complication associated with pregnancy and RSA affects 1% of couples. 129 Although different regions have different definitions, 158 there is a common view of the mechanism of immune-mediated RSA. 132 Immunotherapy has proven to be effective for RSA, including administration of progesterone, cyclosporin A (CsA) and intravenous immunoglobulin (IVIG). [158][159][160][161] Progesterone plays an important role in the establishment and maintenance of pregnancy in an endocrine and immunological manner, which mainly depends on affecting cytokine synthesis and the function of the NK cells. 159,162 It can also treat RSA by restoring several co-inhibitory molecules, including Tim-3, PD-1 and CTLA-4. 163 CsA is a useful immunosuppressant and is used to prevent allograft rejection. In addition to regulating the trophoblast growth and invasion, 164,165 CsA also contributes to maintaining the immunity tolerance at the maternal-foetal interface by upregulating the expression of CTLA-4 and downregulating the levels of CD80, CD86 and CD28. 166   Drug Administration, whereas ipilimumab is pregnancy category C. 176 There is a unique case of conception and pregnancy. Both the mother and the foetus have been successful in the treatment of metastatic melanoma with anti-CTLA-4 and anti PD-1 therapy. 177 In order to understand the full effects of these drugs, long-term follow-up is needed, including other fertility surrogate indicators, such as subsequent pregnancy, abortion, live birth and birth defects. 113 Reproductive safety must be considered when using blocking antibodies during pregnancy.
As mentioned above, the crosstalk between the mother and foetus during pregnancy remains an enigma. With the recognition of innate immune cells recognized, we speculate additional investigations and studies on the immune checkpoint molecules at the maternalfoetal interface might soon be conducted.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.