on the nature and function of human uterine granular lymphocytes

On the nature and function of human uterine granular lymphocytes

Ashley King and Yung Wai Loke

The 'foetal allograft' concept of the maternal-foetal relationship is currently being questioned. This is partly due to the increasing awareness of uterine large granular lymphocytes, a subset of natural killer cells found in the human endometrium at the time of placentation. Analogous cells are present in several species. The possible role of these cells in the regulation of the trophoblast-

maternal interface is discussed here by Ashley King and Yung Wai Loke.

Granulated cells were noted in the pregnant uterine lining (decidua) of many species including rodents (granulated metrial gland (GMG) cells) and humans (endometrial granulocytes) many years ago 1,2. These were sub- sequently identified as lymphoid cells, but detailed characterization by immunohistology and flow cy- tometry have recently shown that human uterine granulated cells have a distinctive surface phenotype reminiscent of CD3- natural killer cells/large granular lymphocytes (NK cells/LGLs) (Refs 3-5, Fig. i and Table 1). However, there are differences between uterine LGLs and peripheral blood NK cells. First, whereas most cir-

culating NK cells are CD16brightCD56 di'11, only about 1% have the decidual phenotype CD16-CD56brig h~ (Ref. 6). Second, the surface density of CD56 (now known as the neural cell adhesion molecule (NCAM)) 7 is over 20 times that found on circulating NK cells (Ref. 8 and Table 2). Third, while the small CD56 bright subset in peripheral blood is agranular, the CD56 bright uterine cells are mostly granulated. These cells account for approximately 70% of bone marrow cells in the decidua during the early stages of placentation 8.

A further characteristic of uterine LGLs is their hor- mone dependence. In human nonpregnant endometrium,

Fig. 1. A frozen section of decidua at eight weeks gestation is stained for CD56. Numerous positive cells can be seen centred around glandular epithelium.

© 1991, Elsevier Science Publishels Ltd, UK, 0167--4919/91/$02.00

Immunology Today 432 Vol 12 No. 12 1991

Table 1. Phenotype of main population of uterine large granular lymphocytes a

Predominant reactivity Cluster differentiation of cell markers (CD number)

Haematopoietic cells CD45 + NK markers CD56 bright++

CD16dim+/- CD57-

Early T-cell markers CD2 + CD7 +

Late T-cell markers CD4- CD8- CD5- CD3-

Integrins CD11a + CD18 +

IL-2R CD25- IL-2Rf3

Activation molecules CD69 + CD45RA +/- HML_I+/-B

aData summarized from Ref. 8. bRef. 42. HML: human placental lactogen.

the number of LGLs varies through the menstrual cycle, with a significant increase in CD56 + cells occurring in the mid-luteal phase, the time at which implantation occurs 4. We consider that this is not due to migration of terminally-differentiated cells into the decidua, as there is extensive proliferation of CD56 + LGLs in utero during the post-ovulatory phase of the menstrual cycle, which continues into early pregnancy 8.

Uterine CD56 bright cells have been shown to exhibit NK-like cytotoxic activity against the K562 cell line, but, like their peripheral blood CD56 bright counterparts 6, the level of killing is lower than that seen with classical NK cellsg, 1°. Decidual LGLs can be induced to proliferate when cultured with interleukin 2 (IL-2) and NK-like killing is then greatly increased 1°,1t. These IL-2-induced responses are presumably mediated via the [3 (p75) chain of the IL-2 receptor (IL-2Rf3), which is expressed by

Table 2. Comparison of the main subsets of CD3- cells in peripheral blood and decidua a

Peripheral blood Decidua CD3- cells CD3- cells

>90% <10%

Phenotype CD56 + CD56 bright+ CD56 bright++ CD16brig ht+ CD16 -/dim+ CD16- CD57 +/- CD57- CD57-

Morphology LGL Agranular Mainly LGL

Naive NK High Low Low activity

Cytokine TNF-~ TNF-c~ ? production IFN-~

aData from Refs 8 and 15. LGL: large granular lymphocytes; TNF-(x: turnout necrosis factor c~; IFN-~/: gamma-interferon.

uterine CD56 + cells (Ref. 12 and King et al., submitted). Although the high-affinity IL-2R receptor (CD25) is expressed on CD56 bright cells in blood 13,14, it is absent from uterine CD56brig ht cells, even after IL-2 stimulation (Ref. 12 and King et al., submitted).

Are uterine granulated lymphocytes NK cells? How, then, are uterine CD56+ cells related to classical

NK cells? They may represent an immature stage of NK- cell differentiation or they could be a distinct NK/LGL subset specific to the uterus (Ref. 15 and Table 2). We favour the second hypothesis. In the uterus, CD56 bright cells proliferate and acquire cytoplasmic granules together with increased surface expression of CD56, but there is no evidence of differentiation to classical NK cells. For example, in vivo, in normal pregnancy, there is no acquisition of CD 16 by decidual LGLs 4,16. The uterus is also the only organ or mucosal surface where CD56 bright cells have been localized. Indeed, even in the uterus itself, these cells are present only in the corpus uteri, and not in the adjacent Fallopian tube or cervix, unless there are ectopic foci of decidualization 2,17. The very small population of agranular CD56brightCD16 - cells in peripheral blood could be migrating from the bone marrow to the uterus which, thus, acts as an extrathymic site for further differentiation. The observation that this subset is more prominent in the peripheral blood of females than in males supports this idea s .

Possible functions of human uterine granular lymphocytes

The functions of uterine LGLs/GMG cells are un- known. Because they are present in such large numbers only during the period in which placentation is established, it is probable that they have an important role to play during early pregnancy.

Human placentation is accompanied by very extensive invasion of uterine tissue by trophoblast cells which infiltrate and destroy the media (muscular wall) of the decidual spiral arteries, converting them from high resistance vessels into distensible sinusoidal tubes that are unresponsive to vasoconstrictive influences and can maintain an adequate blood flow to the fetoplacental unit in all physiological conditions (Ref. 18 and Fig. 2). Normal pregnancy depends on this vascular adaptation by trophoblast, without which a variety of obstetrical problems, such as pre-eclampsia, intra-uterine growth retardation and stillbirth, will result 19. This remarkable phenomenon is not widely appreciated and deserves emphasis. Successful implantation and pregnancy thus depend on a certain degree of trophoblast invasion. Yet, trophoblast over-invasion rarely occurs except in those instances when the placenta implants over areas deficient in decidua, such as in ectopic tubal pregnancies or in the uterus over a previous Caesarian scar tissue 19. These pathological conditions are associated with tubal or uterine rupture and can lead to maternal death. The extent of trophoblast migration, therefore, must be finely controlled by decidua and it is possible that decidual LGLs provide one element of this control.

We propose that the mechanism of this control is by decidual LGLs interacting with an unusual HLA class I molecule expressed by trophoblast, which has now been

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Fig. 2. (a) A histological section of a maternal artery in the placental bed is shown. The normal media (m) is surrounded by trophoblast cells (tr). (b) A more distal section of the same artery shows trophoblast cells (tr) infiltrating and destroying the media (m). (c) A maternal spiral artery in early decidua is stained for human placental lactogen (HPL). The endothelium is replaced by HPL +

trophoblast cells.

established to be a product of the HLA-G locus 2°,21. HLA-G is nonpolymorphic and has a restricted distri- bution, confined to the extravillous trophoblast population invading into decidual tissue and arteries. Villous trophoblast, which is bathed in maternal blood, ex- presses neither MHC class I nor class II antigens. Immunohistological studies whereby extravillous trophoblast was double-stained for a trophoblast marker and MHC class I simultaneously have shown that these cells

are MHC class I negative within the trophoblast columns nearest to the villous core and acquire this antigen only when they migrate outwards towards decidua 22.

Although NK/LGL target structures are not clearly defined, it now seems that multiple monomorphic recep- tors may be involved and that variation of no one cell surface molecule shows a complete correlation with target cell resistance 23. However, in many model systems it has been shown that NK cells preferentially kill target cells with little or no surface expression of MHC molecules. This phenomenon has led to the 'missing self' hypothesis, which states that the absence of self-HLA molecules on target cells is detected by NK cells, rather than the presence of some specific NK recognition molecule 24. The MHC class I molecules may counteract a positive signal to NK cells and, therefore, larger numbers of these molecules would probably be required to inhibit lymphokine-activated killer (LAK) cells.

The 'missing self' hypothesis may explain trophoblast cell survival in maternal decidual tissue. Extravillous trophoblast, by the expression of monomorphic HLA-G, may be seen by maternal NK/LGL effectors as self or neutral and will not, therefore, be killed by uterine NK cells. This will allow the necessary degree of trophoblast invasion to take place for proper establishment of the placenta and its blood supply. The fine tuning of this process will be controlled on the placental side by the level of HLA-G surface expression on trophoblast and on the uterine side by the degree of lymphokine activation of decidual LGLs. Cytokines with potential influence, such as alpha-interferon (IFN-~), IL-2 and transforming growth factor [3 (TGF-~3), have been reported to be present in the uteroplacental interface 2s-28.

It has been possible only recently to study the inter- action between human extravillous trophoblast and decidual LGLs in vitro, with the development of techniques for the isolation of homogeneous populations of these cells 9,29. To date, it has been observed that human first trimester extravillous trophoblast is resistant to lysis by fleshly-isolated decidual LGLs 9 but becomes susceptible when the decidual effectors are stimulated into LAK cells by recombinant IL-2 (Ref. 11). In addition, upregulation of trophoblast surface MHC class I molecules by IFN-7 (Ref. 30) partially protects the human trophoblast from decidual LAK cell killing (authors' unpublished observations). A similar observation has been reported for the susceptibility of the HLA-G-expressing malignant trophoblast cell line JEG-3 to lysis by peripheral blood NK cells 31.

The expression of a monomorphic HLA class ! molecule by trophoblast should also mean that antigen cannot be presented to maternal CD3 ÷ T cells, although these are very sparse in the decidua 4,8. In support of this nonrecognition, it has been shown that JEG-3 chorio- carcinoma cells cannot generate specific cytotoxic or proliferative responses in allogeneic peripheral blood lymphocytes from several donors 31. We have obtained similar results using normal trophoblast and decidual leucocytes (unpublished observations).

Uterine granulated cells in other species Similar granulated uterine lymphoid cells are found in

many other species 32. In particular, GMG cells in the

Immunology Today 434 vot 12 No. 12 1991

mouse, like their human uterine LGL counterpart, also appear to be derived from a lineage distinct from, but relfited to, classical NK cells 33-35. The restraining influence of murine decidua on trophoblast behaviour is well demonstrated by classic experiments of ectopic implantation, where blastocysts transferred into mice kidneys resulted in excessive trophoblast proliferation and con- siderable tissue destruction, while similar blastocysts placed into the decidualized uterus developed nor- mally 36. It is possible that this control is provided by the GMG cells present in decidua. However, there is currently no evidence for the expression of a nonclassical MHC class I antigen analogous to human HLA-G in the murine trophoblast, although such a molecule (the Pa antigen) has been documented in the rat 37. It is, therefore, not clear how GMG cells exert their control on the murine trophoblast. Placental development in different species is highly variable, particularly with respect to the degree of trophoblast invasion required for successful implantation, so that the actual mechanism by which human and murine decidual LGLs interact with their respective trophoblast cells may well differ in detail while maintaining their ultimate function of controlling the extent of trophoblast invasion 32,38. It is also possible that CD3- granulated cells influence other cellular elements in the placental bed, such as epithelial or endothelial cells, afialogous to postulated functions of granulated gut i~ntraepithelial lymphocytes 39.

Conclusion Because the process of mammalian implantation

necessitates varying degrees of invasion into maternal tissues by foetally-derived 'foreign' cells, some protection for the mother must have arisen during the evolutionary development of this form of reproduction. To fulfil this protective role, mammals must have used a defence system which already existed at this early period of evolution. NK cells could be representative of such a primitive system (CD56 ÷ cells have been identified in snails4°). The relationship between the allogeneic placenta and the maternal uterus, therefore, is not governed by currently accepted laws of transplantation biology, in spite of superficial resemblance to an organ graft 41. In- stead, mammalian placentation appears to be based on an older system of immunological recognition which is, as yet, poorly defined.

Ashley King and Yung Wai Loke are at the Dept of Pathol- ogy, Cambridge University, Tennis Court Road, Cambridge CB2 1QP, UK.

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on the nature and function of human uterine granular lymphocytes

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