Trophoblast Research 10:173-180, 1997

T R O P H O B L A S T I N T E R A C T I O N W I T H D E C I D U A L

N K C E L L S I N H U M A N I M P L A N T A T I O N

- A R e v i e w -

Ashley King and Y.W. Loke

Research Group in Human Reproductive Immunobiology Department of Pathology University of Cambridge

Cambridge CB2 1QP United Kingdom

INTRODUCTION

Implantation of the human placenta is accompanied by migration of trophoblast cells from the tips of the anchoring villi into the decidua. The extent of this migration is tightly controlled so that trophoblast cells do not normally penetrate beyond the inner third of the myometr ium where they differentiate into multinucleated placental bed giant cells. The mechanisms of this control are not known. Because these trophoblast cells are fetally-derived and therefore foreign to the mother, it is expected that there may be some immunological recognition by the mother which could influence the migration. For this reason, reproductive immunologists are very interested in the type of immune response which may be generated locally in the uterus against trophoblast. Intriguingly, evidence to date has revealed that this uterine immune response is not the same as that encountered in classical transplantation immunology (Loke and King, 1995). There are fundamental differences occurring in both the trophoblast and the uterus. On the trophoblast side, it is now established that the HLA antigens expressed by trophoblast are not the same as those expressed by other somatic cells. On the uterine side, the mucosa of this organ is populated by cells of the innate immune system, such as Natural Killer (NK) cells rather than T and B lymphocytes characteristic of the specific acquired immune response. Thus, the immunological interaction between human trophoblast and the uterus is likely to be unusual.

Trophoblast Expression Of HLA Class I Antigens

It is now generally accepted that extravillous trophoblast (EVT), the population that migrates into decidua during implantation, expresses the non-classical HLA-G. There is evidence for the presence of both HLA-G mRNA (Yelavarthi et al., 1991; Chumbley et al., 1993) and protein (Kovats et al., 1990; Chumbley et al., 1994) in these cells. Although low levels of HLA-G message is found in a variety of cell types besides trophoblast, such as fetal eye, fetal thymus (Shukla et al., 1990), fetal liver (Houlihan et al., 1992), circulating T and B cells (Kirszenbaum et al., 1994) and even adult skin biopsies (Ulbrecht et al., 1994), recent studies with an HLA-G specific antibody has localized the protein only in EVT (Chumbley et al., 1994). Thus, it seems that expression of the HLA-G antigen is restricted to this trophoblast population. The HLA-G protein exists in several isoforms probably resulting from differential splicing (Ishitani and Geraghty, 1992). In addition, soluble as well as membrane forms have been observed (Fujii et al., 1994).

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The function of HLA-G is unknown. It is relatively non-polymorphic at the genetic and protein level. Although a number of sequence variations in the HLA-G gene which could result in amino-acid substitutions have been reported in different individuals (van der Ven and Ober, 1994), only one of these variations is actually at a functional residue of the pept ide binding groove (Parham, 1995). Therefore, unlike the classical class I genes, polymorphism of HLA-G does not appear to be selected for pept ide-binding diversity which implies that it has probably not evolved for classical T cell interaction. Furthermore, the finding that HLA-G antigen is not expressed in the fetal thymus where T cell education takes place further supports the conclusion that HLA-G is not involved in influencing the T cell repertoire. We believe that HLA-G expressed by EVT functions as a target molecule for decidual Natural Killer (NK) cells rather than T cells (King and Loke, 1991; Loke and King, 1991). However, the HLA-G molecule is capable of binding endogenous nonapept ides in the same way as classical class I molecules. The pept ide may merely enhance the stability of the class I molecule at the cell surface, but the possibility that it can also interact with T cells should not be entirely dismissed.

The potential role of HLA class I antigens in t rophoblast-decidual NK interaction is further complicated by the finding that EVT appears to express another class I molecule besides HLA-G (Grabowska et al., 1990). The identity of this molecule has been much debated, but the accumulative evidence indicates that it is probably HLA-C (King et al., 1996). For a classical class I antigen, HLA-C is rather unusual and differs from HLA-A and -B in having a relatively low surface expression and being less polymorphic. Because of these characteristics, the importance of HLA-C in interacting with T cells has been questioned. Again, as for HLA-G, we believe that HLA-C expressed by EVT is recognized by decidual NK cells (Loke and King, 1995).

Decidual Natural Killer (NK) Cells

The most abundant lymphoid cells in decidua are NK cells which comprise about 70% of the total populat ion with macrophages making up 20% and T lymphocytes 10% (King et al., 1989). There are insignificant numbers of B lymphocytes and granulocytes are absent. The majority of the NK ceils have prominent cytoplasmic granules which have led to their being called Large Granular Lymphocytes (LGL). The number of these NK cells is low in the proliferative stage of the menstrual cycle, gradually increases during the mid-luteal phase and reaches a peak in the late secretory phase. They will show signs of apoptosis a few days before menstruation. However, if pregnancy occurs, their number increases during the early stages of pregnancy, particularly in the decidua basalis and then declines in the second trimester. Immunohistological studies have demonstra ted these NK cells to be in close proximity to the invading trophoblast cells at the implantat ion site. This temporal and spatial association between deciduaI NK cells and EVT suggests a functional interaction between these two cell types.

Phenotypic analysis has reveaied major differences between decidual NK cells and NK cells from peripheral blood, such as the expression of the NK cell marker, CD56 and the FcyRIiI, CD16. In blood, 90% of the NK ceils are CD56 dim CD16brig ht and only 10% are CD56brig ht and CD16 dim. In contrast, in the uterus the majority of NK cells are CD56brig ht CD16-. In addition, CD57, another marker for adult NK cells, is also not expressed by decidual NK cells (Table 1). It would seem, therefore, decidual NK ceils

Trophoblast and NK Cells 175

probably represent a sub-population of the NK cell family which is restricted to the uterus.

Table 1

Characteristics Of NK Cells From Blood, Decidua And Fetal Liver (From: Loke and King, 1995)

Blood Decidua Fetal Liver

NK markers CD56 ~im CD56 br~ ..... CD56 b''~h~ CD16 br~h~ CD16 CD16/'~'~' CD57 ' / CD57 CD57

Cytoplasmic cyCD3 cyCD3 ' cyCD3 ~

Activation markers CD69 CD69" CD69 ~

NK activity High Low Low Respond to high Respond to low Respond to low

dose IL-2 dose IL-2 dose IL-2

Cytokine GM-CSF GM-CSF" GM-CSF ~ Production CSF-1 CSF-1 ~ CSF-I?

The ontogeny of decidual NK cells is unclear. Two possible differentiation pathways can be proposed (Loke and King, 1995). Decidual NK cells could have arisen from a common NK cell progenitor as those in blood, but have subsequently undergone tissue-specific differentiation under the influence of some uterine signal resulting in a unique phenotype. Alternatively, decidual NK may represent a distinct NK cell population which has split off early in its differentiation pathway from that in blood to settle in the uterus.

The variation in number of NK cells over the menstrual cycle and in pregnancy suggests their recruitment/maintenance is likely to be under hormonal control but the identity of the stimulus is not known. Decidual NK cells do not express estrogen, progesterone or prolactin receptors. They do express the intermediate affinity p75 and the high affinity p55 IL-2R (Nishikawa et al., 1991), and they have been observed to proliferate in the presence of IL-2 in vitro (King et al., 1992). However, IL-2 has not been localized in decidua in vivo (Loke and King 1995), and also IL-2 transforms decidual NK cells into powerful lymphokine-activated killer (LAK) cells which are capable of killing trophoblast cells in vitro (King and Loke, 1990), so this cytokine is unlikely to have a physiological role to play. The search is now on for a cytokine which can induce proliferation in decidual NK cells without transforming them into LAK cells in vitro, and which is demonstrable in uterine tissues in vivo.

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Trophoblast-Decidual NK Cell Interaction

Although we have proposed that trophoblast invasion of the uterus during the process of implantation is controlled by decidual NK cells interacting with trophoblast HLA-G/HLA-C, elucidation of the exact mechanism of this interaction has proved elusive. This is because compared to T cells little is known about NK cell biology, NK target molecules, the NK cell receptors and the outcome of any interaction between NK cells and target cells (Figure 1).

J l ~ Cytotoxicity

T ~ ~ f C~:tiPr:d ucti~

I I Tce"reeeptor ~ Peptide

s I molecule

? ?

? ?

Cytotoxicity

Cytokine production

Figure 1. Diagram showing that the T cell receptors and its associated target ligand are known, but the equivalent structures for NK cells have not been established. (From: Loke and King, 1995)

Trophoblast and NK Cells 177

Maternal-Fetal Interaction

Uterine NK Cells ~ Extravillous trophoblast

negative/ sign7 \ posi,ive HLA-G + and ?HLA-C + negative/ ignal

Killing Cytokine production

Figure 2. A hypothetical model of the possible outcome of interaction between uterine NK cells and extravillous trophoblast at the implantation site. (From: Loke and King, 1995)

However, it is now generally accepted that NK cells preferentially kill target cells which have low or absent HLA class I antigen expression. This observation had led to the formulation of the 'missing-self' hypothesis which postulated that NK cells eliminated cells lacking class I molecules in contrast to T cells which kill cells bearing foreign class I antigens. Thus the presence of class I molecules on target cells prevents NK-mediated lysis (Ljunggren and K~irre, 1990). The situation has since become more complex as it is now apparent that NK cells can discriminate between different class I alleles, although they do not detect fine polymorphic differences like T cells. Instead, NK cells appear to recognize a public, perhaps primordial , polymorphism in class I molecules. A family of human NK receptors which do recognize HLA class I antigens have recently been identified, the p58 (NKAT) family of receptors for Cw3/Cw4 and related alleles (Gumperz and Parham, 1995). There are now designated Killer Inhibitory Receptors (KIR). Each NK cell can express several different receptors, both KIR which recognize class I molecules, and other receptors of different molecular structure which possibly use target l igands such as oligosaccharides. It seems that this is the way receptor diversity is generated in NK cells rather than by somatic recombination of a single receptor as used by T and B cells. Expression of different combinations of these receptors on each NK cell will determine its repertoire (Yokoyama, 1995). Furthermore, the NK repertoire in blood NK cells varies between different individuals with the intriguing observation that KIR which have specificity for non-self class I molecules may occur (Gumperz and Parham, 1995). These recent findings raise the fascinating possibility that maternal allorecognition of the fetus does occur, but it is mediated in a completely novel manner by NK cells rather than a classical allograft reaction mediated by T cells.

In the context of interaction with trophoblast, the expression of HLA-G/HLA-C by trophoblast could influence their susceptibility to decidual NK cell lysis. Exposure of human trophoblast cells to exogenous IFN- 7, a cytokine which upregulates class I expression, has been observed to protect trophoblast cells against lysis by IL-2-stimulated decidual NK cells (King and Loke, 1993). Similarly, HLA-G transfected into class I-

178 King and Loke

deficient cell lines was found to provide some protection from lysis by freshly isolated decidual NK effectors compared to the parental cell lh~e (Chumbley et al., 1994).

The addit ional possibility that class I signals transmitted to decidual NK cells can affect not only cytotoxicity but also influence other NK cell functions such as cytokine product ion would need to be explored (Figure 2). Decidual NK cells are known to produce a variety of cytokines and trophoblast expresses appropriate receptors for many of these cytokines. Thus a potential cytokine network may be in place at the implantation site by which decidual NK ceils influence trophoblast behavior.

CONCLUSION

From this brief review, it can be seen that the immunological relationship between the implanting placenta and the maternal uterus is not governed by the laws of classical transplantation immunology. Instead, it seems to involve a more primitive defense system whose mechanism of 'self' and 'non-self' recognition and the resultant reactions invoked, are more akin to that observed between unrelated invertebrates than between vertebrate allograft and host. This observation has completely altered our conceptual view of the immunology of reproduction.

ACKNOWLEDGEMENTS

AK is in receipt of the Meres Senior Studentship for Medical Research of St. John's College, Cambridge. YWL is funded by the Medical Research Council and by the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization.

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Fujii, T., Ishitani, A. and Geraghty, D.E. (1994) A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4. J. ImmunoI. 153, 5516-5524.

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