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Recognition of Trophoblast HLA Class I Molecules by Decidual NK Cell Receptors—A Review
Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S81–S85 doi:10.1053/plac.1999.0520, available online at http://www.idealibrary.com on
IMMUNOLOGY Recognition of Trophoblast HLA Class I Molecules by Decidual NK Cell Receptors—A Review A. Kinga, S. E. Hiby, L. Gardner, S. Joseph, J. M. Bowen, S. Verma, T. D. Burrows and Y. W. Loke Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, UK Paper accepted 16 December 1999
During placentation the extravillous trophoblast (EVT) cells migrate through the decidua towards the maternal spiral arteries. The walls of the arteries are then destroyed by trophoblast resulting in an increased blood flow to the fetus. These EVT express HLA-G, HLA-E and HLA-C, an unusual combination of two non-classical and one classical MHC class I molecules. The decidua is infiltrated by distinctive uterine natural killer (NK) cells during the time of trophoblast invasion. These cells express a variety of receptors (CD94/NKG2, KIR and ILT) which are known to recognize HLA class I molecules. There is, therefore, a mechanism for molecular recognition of the placental trophoblast cells. The possible functional consequences of this uterine NK cell–trophoblast interactions are uncertain. One possible result is in an altered NK cell cytokine profile which modulates the invasive proclivity of the EVT. In this way placentation could be controlled. 2000 IFPA and Harcourt Publishers Ltd Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S81–S85
TROPHOBLAST INVASION INTO THE UTERUS The placental extravillous trophoblast cells (EVT) which infiltrate into the maternal uterine mucosa (decidua) have an essential function in tapping into the maternal blood supply to achieve the increased conductance needed for normal fetal growth and development. Failure of vascular transformation by trophoblast is the underlying primary defect in common disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction (IUGR). EVT intermingle with a variety of maternal leukocytes at the implantation site in decidua, notably the abundant natural killer (NK) cells together with macrophages and a few / CD3 T cells (Loke and King, 1995). Therefore, the major histocompatibility complex (MHC) status of EVT is of importance in recognition of the semiallogeneic fetus by the mother. As a result of this interaction between maternal uterine leukocytes and fetal EVT the extent of vascular transformation and trophoblast invasion may be regulated.
a
To whom correspondence should be addressed at: Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. Fax: +44 1223 333727; E-mail: [email protected]
0143–4004/00/0A00S81+05 $35.00/0
EXPRESSION OF HLA CLASS I MOLECULES BY EVT EVT express no HLA class II molecules but do express an unusual combination of HLA class I molecules HLA-C, HLA-E and HLA-G. Thus, one classical and two non-classical HLA class I molecules are expressed. HLA-G has always attracted most attention because of its unusual characteristics: restricted expression to EVT, negligible polymorphism and the existence of splice variants (Hiby et al., 1999). HLA-C has been surprisingly neglected, although we and others now have definitive evidence that it is expressed on the trophoblast cell surface both as conventional 2-m bound forms and as free heavy chains (King et al., 1996; King et al., 2000). Unlike HLA-G, HLA-C surface expression is upregulated by IFN-. Immunohistology indicates that the pattern of HLA-C expression by trophoblast overlaps that of HLA-G. Importantly, both paternal as well as maternal alleles are transcribed. Therefore, in terms of allorecognition in the context of reproduction, trophoblast paternally-derived HLA-C molecules are important to consider. HLA-E molecules are only found on the cell surface if signal peptides derived from certain other HLA class I molecules are present in the ER (Braud, Jones and McMichael, 1997). HLA-G and HLA-C signal peptides both bind efficiently to the HLA-E peptide-binding groove. By immunoprecipitation of trophoblast proteins with W6/32 and subsequent analysis by IEF we have now 2000 IFPA and Harcourt Publishers Ltd
Placenta (2000), Vol. 21, Supplement A, Trophoblast Research, Vol. 14
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Killer cell immunoglobulin-like receptors
KIR CD94
Immunoglobulin-like transcripts
ILT
C-type lectin-like molecules
CD94/NKG2
CD94
NKG2A
NKG2C
DAP12 +
Figure 1. Receptors for HLA class I molecules expressed on human NK cells.
–
ITAM
ITIM
demonstrated that HLA-E is expressed on the trophoblast cell surface (King et al., submitted).
Activating
Inhibitory
Figure 2. Structures of CD94/NKG2.
NK CELL RECOGNITION OF HLA CLASS I MOLECULES The main effector functions of NK cells in vivo are cytotoxicity and cytokine production. Both these activities are regulated by receptors for MHC class I antigens. These NK cell receptors were discovered following the observation that NK cells could eliminate tumour cells which lacked MHC class I proteins (Ljunggren and Karre, 1990). The NK cell receptors which bind HLA class I molecules in humans belong to three structurally distinct families: CD94/NKG2 heterodimers, killer cell Ig-like receptors (KIR) and Ig-like transcripts (ILT) (Lanier, 1998; Long, 1999) (Figure 1). The CD94/NKG2 receptor is a member of the C type lectin superfamily and binds to the non-classical HLA-E molecule (Braud et al., 1998). The invariant CD94 associates with members of the small NKG2 family to form heterodimers. Whilst CD94/NKG2A provides an inhibitory signal preventing lysis, in contrast, CD94/NKG2C is activating. This is due to variation in the cytoplasmic domains of the NKG2 molecules. Inhibitory NKG2A has a longer tail containing paired immunoreceptor tyrosine based inhibition motifs (ITIM). NKG2C lacks ITIM and has a charged residue in the transmembrane region through which it associates with a signalling molecule, DAP-12, which contains an immunoreceptor tyrosine based activation motif (ITAM) (Long, 1999) (Figure 2). The inhibitory CD94/NKG2A receptor has higher affinity for HLA-E than the activating CD94/NKG2C. Furthermore, the binding affinity is also dependent on the sequence of the bound HLA-derived signal peptide. An HLA-E molecule loaded with the HLA-G leader sequencederived peptide binds both CD94/NKG2A and CD94/ NKG2C with the highest affinity of all class I-derived signal peptides (Vale´ s-Go´ mez et al., 1999). The KIR are encoded by a family of about 10 genes. The extracellular domains may have two (KIR2D) or three (KIR3D) Ig-like domains (Figure 3). It is the KIR2D receptors which predominantly recognize HLA-C. KIR2D discriminates between two groups of HLA-C allotypes distinguished by the presence of a dimorphism at positions 77 and 80 of the 1 domain (Long, 1999). There are also some structurally divergent KIR2D molecules, for example, KIR2DL4, whose ligands
γ1
γ1
γ2
γ2
γ2
γ2
γ3
γ3
γ3
γ3
γ1 γ3
+
– +
–
+
K ITIM
R
ITAM DAP12
Inhibitory KIR
ITAM
ITIM
DAP12
Activating KIR
KIR2DL4
Figure 3. Structures of KIR.
are still being elucidated. Like the CD94/NKG2 receptors, KIR also exhibit variation in the structures of the cytoplasmic domains resulting in either inhibitory or activating forms. There are members with long tails containing ITIM (KIR2DL) and those with short tails which associate with ITAM-containing DAP-12 (KIR2DS). One other feature of KIR is of interest. NK cell receptors are expressed on overlapping subsets of NK cells and one NK cell simultaneously expresses either two or more KIR and/or CD94/ NKG2 and ILT. There is always at least one inhibitory receptor for self HLA class I molecules expressed but they also can express seemingly useless KIR, whose ligand is for non-self (Lanier, 1998). These receptors could be important in reproduction which is the only physiological situation where NK cells come into contact with non-self. The ILT family consists of at least eight members with different numbers of Ig domains and again there are activating and inhibitory forms. However, unlike KIR, whose expression is almost entirely restricted to NK cells, ILT are found predominantly on cells of the monocytic lineage, B cells and also on NK and T cell subsets. Only two members of the ILT family (ILT2 and ILT4) have so far been found to bind to a broad range of HLA class I molecules including HLA-G (Long, 1999).
King et al.: Trophoblast HLAI Molecules Recognition
UTERINE NK CELL RECEPTORS FOR TROPHOBLAST HLA CLASS I MOLECULES The NK cells in the uterine mucosa are a distinctive cell type (CD56bright, CD16) whose relationship with circulating CD56 + NK cells is unknown (King et al., 1998). Once it became obvious that NK cells as well as T cells were capable of allorecognition, the possibility that maternal uterine NK cells used this allorecognition system to sense the presence of the semi-allogeneic fetus was raised (King and Loke, 1991). The close association between these unusual NK cells and EVT also suggested the existence of NK cell receptors which could potentially recognize trophoblast HLA-G, HLA-C and HLA-E. This NK cell/trophoblast interaction could result in modulation of the invasiveness of the EVT and, therefore, the extent of vascular transformation and the maternal blood flow to the fetus.
HLA-E Virtually all decidual CD56bright decidual NK cells express CD94/NKG2A at five times a greater intensity than blood NK cells as measured by flow cytometry. The coexpression of CD94/NKG2C cannot be determined as there are no Mabs available to the NKG2C molecule. However, NKG2C transcripts are present in FACS-sorted CD56bright cells by RT-PCR (unpublished). We have recently demonstrated that more than 95 per cent of decidual NK cells will bind to soluble HLA-E tetrameric complexes and that this binding is inhibited by Mabs to CD94 (King et al., submitted). Therefore, HLA-E could act as a sentinel molecule sensitive to levels of expression of other HLA class I molecules. In addition, the particularly high affinity for HLA-E molecules with the HLA-G-derived leader sequence bound in its groove means that maternal uterine NK cells can potentially sense and respond to the HLA-G + fetal trophoblast.
HLA-C KIR2D molecules specific for HLA-C are expressed by decidual NK cells (Verma et al., 1997; Hiby et al., 1997). These KIR have been demonstrated by RT-PCR and sequencing of CD56bright cells sorted to a high degree of purity by flow cytometry, by immunostaining of tissue sections and by flow cytometric analysis of dissociated decidual leukocytes. No unique KIR sequences were found which had not been isolated from blood NK cells, so the potential repertoire used by maternal uterine NK cells is the same as for blood NK cells. However, using Mabs GL183 and EB6, we found that a higher proportion of CD56bright NK cells were GL183 + and/or EB6 + compared to peripheral blood NK cells analysed at the same time from a pregnant individual. (Mabs GL183 and EB6 are specific for KIR2D receptors which bind to the two groups of HLA-C allotypes). At 8–10 weeks gestation, 50–80 per cent of
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decidual NK cells were reactive with either or both Mabs compared to only 5–20 per cent in peripheral blood CD56 + cells (Verma et al., 1997). Therefore, the maternal uterine NK cells appear skewed towards HLA-C recognition compared to those in peripheral blood.
HLA-G A receptor expressed by decidual NK cells which is specific for HLA-G has remained intriguingly elusive. Early reports that members of the KIR family specific for HLA-C or HLA-B could also bind HLA-G were not subsequently confirmed. It was then thought that CD94/NKG2 mediated HLA-G recognition, but these data are now explained by the coexpression of HLA-E on the target cells used in the cytotoxicity assays (Long, 1999). Two other receptors are now candidates for the HLA-G receptor. Members of the ILT2 family were found to bind to a wide range of HLA class I molecules, including HLA-G in cytotoxicity assays, and by direct binding of soluble ILT fusion proteins to cells transfected with single HLA class I molecules (Long, 1999). When soluble tetrameric complexes of HLA-G were tested for binding to peripheral blood mononuclear cells it was found that only a subset of blood monocytes were stained and there was no binding at all to NK cells. Binding of the HLA-G tetramers was found to be mediated by ILT receptors, mainly by ILT4 with some contribution by ILT2, but only when the latter was expressed at high levels (Allan et al., 1999). ILT2 is expressed by 20–25 per cent of decidual NK cells in all women and both ILT2 and ILT4 are present on all decidual macrophages (Verma et al., 1997 and unpublished). However, the broad specificity of ILT2 and ILT4 for many HLA class I molecules would suggest that HLA-G is not providing a unique ‘fetal’ signal to maternal leukocytes. These results suggest that HLA-G could modulate maternal macrophage, rather than NK cell, behaviour at the maternal– fetal interface. Recently, there is evidence that one of the orphan (i.e. with no recognizable ligand) KIR, KIR2DL4, will bind to HLA-G (Lanier, 1999; Rajagopalan and Long, 1999; Ponte et al., 1999) (Figure 3). When expressed at high levels in an NK cell line, KIR2DL4 inhibited killing of a target cell transfected with HLA-G. In addition, soluble KIR2DL4 fusion proteins bound to HLA-G transfectants, but not to similar cells transfected with other HLA class I molecules. KIR2DL4 is different from other KIR in expression and structure. Amongst its unique structural features is the combination of a cytoplasmic domain containing a single ITIM with a charged residue in the transmembrane region. This suggests it might be ‘schizophrenic’ in function, having both inhibitory ITIM functions and also the ability to associate with another activating molecule. The expression pattern is also of interest, as all other KIR are expressed on overlapping NK cell subsets. Whilst one report has described expression on all NK cells (Rajagopalan and Long, 1999), another has only found a subset of decidual
S84
NK cells from early pregnancy are KIR2DL4 + whilst all blood NK cells are negative (Ponte et al., 1999). Development of more reagents should resolve this issue and lead to investigation of the functional consequences of KIR2DL4 interaction with HLA-G in the decidua basalis. The challenge now is to reconcile all these observations. It is clearly important to establish whether HLA-G is predominantly a target recognition molecule for either decidual NK cells and/or macrophages. The binding assay using the HLA-G tetrameric complexes demonstrated no staining of peripheral blood NK cells. In contrast, there was binding of KIR2DL4 fusion proteins to HLA-G transfectants. The possible explanations to explain these conflicting results are as follows: (1) Although HLA-G tetramers did bind to ILT2 transfectants this only occurred if ILT2 was expressed at high levels. The expression levels of ILT2 normally on NK cells may be too low to show detectable binding with the HLA-G tetramers, but this interaction could still be functionally important; (2) The HLA-G tetramers were refolded in vitro around a self-peptide. Whilst KIR recognition is dependent on the sequence at positions 7 and 8 of the bound peptide, ILT recognition is independent of the peptide sequence. Perhaps refolding of the HLA-G protein around a variety of peptides found from elution studies to bind to HLA-G would give different results; (3) The HLA-G protein used to form the tetrameric complexes was prepared in Escherichia coli and would, therefore, not be glycosylated. HLA-G is much more heavily glycosylated when expressed by normal trophoblast compared to JEG-3 choriocarcinoma cells or HLA-G transfectants (McMaster and Fisher, 1998). There is one glycosylation site on HLA class I molecules which is at position 86 on the 1 domain close to the KIR recognition site (77–83). There is still no evidence that glycosylation influences human NK cell recognition despite the suspicions raised by the possession of a carbohydaterecognition domain on the C-type lectin NK cell receptors (Braud et al., 1998). However, MHC-linked carbohydrates may influence NK cell binding in the mouse.
FUNCTIONAL IMPLICATIONS The discovery of NK cell receptors for HLA class I molecules has depended heavily on cytotoxicity assays using target cells transfected with various class I molecules. The use of phenotypically well-defined peripheral blood NK cell clones expressing individual NK cell receptors and specific Mabs has allowed dissection of individual NK cell receptor-HLA class I ligand interactions which either inhibit or activate the NK cell. Direct binding assays using soluble fusion proteins of KIR, ILT and CD94/NKG2 molecules have also been used and the recent development of soluble HLA tetramers has been a
Placenta (2000), Vol. 21, Supplement A, Trophoblast Research, Vol. 14
significant technical advance. There is still difficulty, however, in extrapolating these in vitro assays to the in vivo situation at the implantation site. For example, there are practical and ethical difficulties in generating decidual NK cell clones. Unusual configurations and the glycosylation of HLA class I molecules on trophoblast also means that B cell transfectants are not ideal targets. We have used polyclonal decidual NK cells isolated separately from different individuals to see what the overall effect of recognition of different HLA class I molecules is on cytotoxicity. The high levels of CD94/NKG2A expression do mediate inhibition of killing of decidual NK cells when exposed to target cells expressing HLA-E, and this inhibition is partially reversed in the presence of anti-CD94 Mabs (King et al., submitted). Thus, even if there does turn out to be coexpression of CD94/NKG2C on all the cells, the dominant effect on killing is inhibitory. Trophoblasts may not be the only cells in the decidua basalis which need to survive. NK cells are not generally a tissue cell and are found mainly in blood and, to a lesser extent, in the spleen and bone marrow. There must be fail-safe mechanisms to prevent lysis of the surrounding autologous maternal cells and this may be why CD94/NKG2 is upregulated in the decidua. Therefore, HLA-E could be the survival molecule for all these tissue cells. There is also functional evidence that killing by NK clones derived from peripheral blood or polyclonal decidual NK cells is inhibited by HLA-G. This interaction appears dependent on HLA-G rather than HLA-E, because Mabs specific for HLA-G which have no cross-reactivity to HLA-E partially reverse the inhibition (Navarro, 1999; King et al., submitted). Decidual NK cells will also be able to sense the HLA-G expression by trophoblast through the presentation of the HLA-G leader sequence by HLA-E, and by the expression of ILT2 on a minor subset which binds to HLA-G at low affinity. Because HLA-G is monomorphic it could be viewed as an invariant ‘fetal’ signal signifying that pregnancy has occurred. In contrast, the addition of the anti-KIR Mabs, GL183 and EB6, to cytotoxicity assays using HLA-C transfectants has no effect on killing, despite the high proportion of cells staining with these Mabs. As with CD94/NKG2, we are still unsure whether these KIR are predominantly inhibitory forms or whether activating forms are also abundant. EB6 and GL183 cannot distinguish between these forms but both are transcribed (Hiby et al., 1997). The interest of HLA-C lies in its polymorphism which leads to a potentially different fetal HLA-C from the mother’s HLA-C, and varies in different pregnancies. There is also variability on the maternal side as the KIR genotype differs between individuals. Therefore, the outcome of each pregnancy is different, depending on the maternal NK cell genotype and the paternal HLA-C allotype expressed in the fetus. Because of our original hypothesis that trophoblast survival depended on the expression of the non-classical HLA-G molecule (King and Loke, 1991), we have also performed these cytotoxicity assays using normal trophoblast as target cells in the presence of Mabs to HLA class I molecules, HLA-G,
King et al.: Trophoblast HLAI Molecules Recognition
HLA-E
CD94/NKG2
HLA-C
KIR
HLA-G
ILT4 (+ILT2) KIR2DL4
Figure 4. Decidual NK cell recognition of trophoblast HLA class I molecules.
CD94/NKG2, ILT2 and KIR. We have not been able to detect any lysis of trophoblast, in keeping with another report using JEG-3 cells and blood NK cells (King et al., submitted; Avril et al., 1999). This raises the question of what other effector functions may be triggered by NK cell recognition of trophoblast. The role of NK cells in providing defence against viral infection is widely known but, interestingly, in vivo there is only firm evidence that this is mediated by NK cell cytokines and not by cytotoxicity (Biron et al., 1999). Similarly, in hybrid resistance in mice, an experimental system of NK cell-mediated allorecognition, it is failure of engraftment of the donor cells due to lack of beneficial NK cell cytokines which leads to apparent ‘rejection’ (Aguila and Weissman, 1996). Thus, the focus now should probably be on the cytokines produced by uterine NK cells in response to binding to trophoblast HLA class I molecules. Despite the lack of evidence to show how NK cells influence EVT behaviour, these recent results have, for the first time, provided molecular mechanisms to explain how the mother might recognize the semi-allogeneic fetus (Figure 4).
REFERENCES Aguila HL & Weissman IL (1996) Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells. Blood, 87, 1225–1231. Allan DSJ, Colonna M, Lanier LL, Churakova TD, Abrams JS, Ellis SA, McMichael AJ & Braud VM (1999) Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells. J Exp Med, 189, 1149–1155. Avril T, Jarousseau AC, Watier H, Boucrat J, Le Bouteiller P, Bardos P & Thibault G (1999) Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism. J Immunol, 162, 5902– 5909. Biron CA, Nguyen KB, Pien GC, Cousens LP & Salazar-Mather TP (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines. Ann Rev Immunol, 17, 189–220. Braud VM, Jones EY & McMichael A (1997) The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol, 27, 1164–1169.
S85 Braud VM, Allan DSJ, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, Lanier LL & McMichael AJ (1998) HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391, 795–799. Hiby SE, King A, Sharkey AM & Loke YW (1997) Human uterine NK cells have a similar repertoire of Killer Inhibitory and Activatory receptors to those found in blood, as demonstrated by RT-PCR and sequencing. Mol Immunol, 34, 419–430. Hiby SE, King A, Sharkey A & Loke YW (1999) Molecular studies of trophoblast HLA-G: polymorphism, isoforms, imprinting and expression in pre-implantation embryo. Tissue Antigens, 53, 1–13. King A & Loke YW (1991) On the nature and function of human uterine granular lymphocytes. Immunol Today, 12, 432–435. King A, Burrows TD, Hiby SE, Joseph S, Verma S, Lim PB, Gardner L, Bowen JM, Ziegler A, Uchanska-Ziegler B, Le Bouteiller P & Loke YW. (2000) Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta, in press. King A, Allan DSJ, Joseph S, Verma S, Hiby SE, Bowen JM, McMichael AJ, Braud VM & Loke YW. HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Submitted. King A, Boocock C, Sharkey A, Gardner L, Beretta A, Siccardi AG & Loke YW (1996) Evidence for the expression of HLA-C class I mRNA and protein by human first trimester trophoblast. J Immunol, 156, 2068–2076. King A, Burrows T, Verma S, Hiby SE & Loke YW (1998) Human uterine lymphocytes. Hum Reprod Update, 4, 480–485. Lanier LL (1998) NK cell receptors. Ann Rev Immunol, 16, 359–393. Lanier LL (1999) Natural killer cells fertile with receptors for HLA-G? Proc Nat Acad Sci USA, 96, 5343–5345. Ljunggren H-G & Ka¨rre K (1990) In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today, 11, 237–244. Loke YW & King A (1995) Human Implantation: Cell Biology and Immunology. Cambridge: Cambridge University Press. Long EO (1999) Regulation of immune responses through inhibitory receptors. Ann Rev Immunol, 17, 875–904. McMaster MT & Fisher SJ (1998) The fetal transplant: is HLA-G important? In Fertility and Reproductive Medicine (Eds) Kempers RD, Cohen J, Haney AF & Younger JB, pp. 661–678. Amsterdam: Elsevier Science B.V. Navarro F, Llano M, Bellon T, Colonna M, Geraghty DE & LopezBotet M (1999) The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognise HLA-G1 and HLA-E molecules co-expressed on target cells. Eur J Immunol, 29, 277–283. Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G, Vitale C, Bertone S, Moretta A, Moretta L & Mingari MC (1999) Inhibitory receptors sensing HLA-G1 molecules in pregnancy: deciduaassociated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proc Nat Acad Sci USA, 96, 5674–5679. Rajagopalan S & Long EO (1997) The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)Cw4 exhibits peptide selectivity. J Exp Med, 185, 1523–1528. Rajagopalan S & Long EO (1999) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J Exp Med, 189, 1093–1100. Vale´s-Go´mez M, Reyburn HT, Erskine RA, Lo´pez-Botet M & Strominger JL (1999) Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. EMBO J, 18, 4250–4260. Verma S, King A & Loke YW (1997) Expression of killer-cell inhibitory receptors (KIR) on human uterine NK cells. Eur J Immunol, 27, 979–983.
IMMUNOLOGY Recognition of Trophoblast HLA Class I Molecules by Decidual NK Cell Receptors—A Review A. Kinga, S. E. Hiby, L. Gardner, S. Joseph, J. M. Bowen, S. Verma, T. D. Burrows and Y. W. Loke Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, UK Paper accepted 16 December 1999
During placentation the extravillous trophoblast (EVT) cells migrate through the decidua towards the maternal spiral arteries. The walls of the arteries are then destroyed by trophoblast resulting in an increased blood flow to the fetus. These EVT express HLA-G, HLA-E and HLA-C, an unusual combination of two non-classical and one classical MHC class I molecules. The decidua is infiltrated by distinctive uterine natural killer (NK) cells during the time of trophoblast invasion. These cells express a variety of receptors (CD94/NKG2, KIR and ILT) which are known to recognize HLA class I molecules. There is, therefore, a mechanism for molecular recognition of the placental trophoblast cells. The possible functional consequences of this uterine NK cell–trophoblast interactions are uncertain. One possible result is in an altered NK cell cytokine profile which modulates the invasive proclivity of the EVT. In this way placentation could be controlled. 2000 IFPA and Harcourt Publishers Ltd Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S81–S85
TROPHOBLAST INVASION INTO THE UTERUS The placental extravillous trophoblast cells (EVT) which infiltrate into the maternal uterine mucosa (decidua) have an essential function in tapping into the maternal blood supply to achieve the increased conductance needed for normal fetal growth and development. Failure of vascular transformation by trophoblast is the underlying primary defect in common disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction (IUGR). EVT intermingle with a variety of maternal leukocytes at the implantation site in decidua, notably the abundant natural killer (NK) cells together with macrophages and a few / CD3 T cells (Loke and King, 1995). Therefore, the major histocompatibility complex (MHC) status of EVT is of importance in recognition of the semiallogeneic fetus by the mother. As a result of this interaction between maternal uterine leukocytes and fetal EVT the extent of vascular transformation and trophoblast invasion may be regulated.
a
To whom correspondence should be addressed at: Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. Fax: +44 1223 333727; E-mail: [email protected]
0143–4004/00/0A00S81+05 $35.00/0
EXPRESSION OF HLA CLASS I MOLECULES BY EVT EVT express no HLA class II molecules but do express an unusual combination of HLA class I molecules HLA-C, HLA-E and HLA-G. Thus, one classical and two non-classical HLA class I molecules are expressed. HLA-G has always attracted most attention because of its unusual characteristics: restricted expression to EVT, negligible polymorphism and the existence of splice variants (Hiby et al., 1999). HLA-C has been surprisingly neglected, although we and others now have definitive evidence that it is expressed on the trophoblast cell surface both as conventional 2-m bound forms and as free heavy chains (King et al., 1996; King et al., 2000). Unlike HLA-G, HLA-C surface expression is upregulated by IFN-. Immunohistology indicates that the pattern of HLA-C expression by trophoblast overlaps that of HLA-G. Importantly, both paternal as well as maternal alleles are transcribed. Therefore, in terms of allorecognition in the context of reproduction, trophoblast paternally-derived HLA-C molecules are important to consider. HLA-E molecules are only found on the cell surface if signal peptides derived from certain other HLA class I molecules are present in the ER (Braud, Jones and McMichael, 1997). HLA-G and HLA-C signal peptides both bind efficiently to the HLA-E peptide-binding groove. By immunoprecipitation of trophoblast proteins with W6/32 and subsequent analysis by IEF we have now 2000 IFPA and Harcourt Publishers Ltd
Placenta (2000), Vol. 21, Supplement A, Trophoblast Research, Vol. 14
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Killer cell immunoglobulin-like receptors
KIR CD94
Immunoglobulin-like transcripts
ILT
C-type lectin-like molecules
CD94/NKG2
CD94
NKG2A
NKG2C
DAP12 +
Figure 1. Receptors for HLA class I molecules expressed on human NK cells.
–
ITAM
ITIM
demonstrated that HLA-E is expressed on the trophoblast cell surface (King et al., submitted).
Activating
Inhibitory
Figure 2. Structures of CD94/NKG2.
NK CELL RECOGNITION OF HLA CLASS I MOLECULES The main effector functions of NK cells in vivo are cytotoxicity and cytokine production. Both these activities are regulated by receptors for MHC class I antigens. These NK cell receptors were discovered following the observation that NK cells could eliminate tumour cells which lacked MHC class I proteins (Ljunggren and Karre, 1990). The NK cell receptors which bind HLA class I molecules in humans belong to three structurally distinct families: CD94/NKG2 heterodimers, killer cell Ig-like receptors (KIR) and Ig-like transcripts (ILT) (Lanier, 1998; Long, 1999) (Figure 1). The CD94/NKG2 receptor is a member of the C type lectin superfamily and binds to the non-classical HLA-E molecule (Braud et al., 1998). The invariant CD94 associates with members of the small NKG2 family to form heterodimers. Whilst CD94/NKG2A provides an inhibitory signal preventing lysis, in contrast, CD94/NKG2C is activating. This is due to variation in the cytoplasmic domains of the NKG2 molecules. Inhibitory NKG2A has a longer tail containing paired immunoreceptor tyrosine based inhibition motifs (ITIM). NKG2C lacks ITIM and has a charged residue in the transmembrane region through which it associates with a signalling molecule, DAP-12, which contains an immunoreceptor tyrosine based activation motif (ITAM) (Long, 1999) (Figure 2). The inhibitory CD94/NKG2A receptor has higher affinity for HLA-E than the activating CD94/NKG2C. Furthermore, the binding affinity is also dependent on the sequence of the bound HLA-derived signal peptide. An HLA-E molecule loaded with the HLA-G leader sequencederived peptide binds both CD94/NKG2A and CD94/ NKG2C with the highest affinity of all class I-derived signal peptides (Vale´ s-Go´ mez et al., 1999). The KIR are encoded by a family of about 10 genes. The extracellular domains may have two (KIR2D) or three (KIR3D) Ig-like domains (Figure 3). It is the KIR2D receptors which predominantly recognize HLA-C. KIR2D discriminates between two groups of HLA-C allotypes distinguished by the presence of a dimorphism at positions 77 and 80 of the 1 domain (Long, 1999). There are also some structurally divergent KIR2D molecules, for example, KIR2DL4, whose ligands
γ1
γ1
γ2
γ2
γ2
γ2
γ3
γ3
γ3
γ3
γ1 γ3
+
– +
–
+
K ITIM
R
ITAM DAP12
Inhibitory KIR
ITAM
ITIM
DAP12
Activating KIR
KIR2DL4
Figure 3. Structures of KIR.
are still being elucidated. Like the CD94/NKG2 receptors, KIR also exhibit variation in the structures of the cytoplasmic domains resulting in either inhibitory or activating forms. There are members with long tails containing ITIM (KIR2DL) and those with short tails which associate with ITAM-containing DAP-12 (KIR2DS). One other feature of KIR is of interest. NK cell receptors are expressed on overlapping subsets of NK cells and one NK cell simultaneously expresses either two or more KIR and/or CD94/ NKG2 and ILT. There is always at least one inhibitory receptor for self HLA class I molecules expressed but they also can express seemingly useless KIR, whose ligand is for non-self (Lanier, 1998). These receptors could be important in reproduction which is the only physiological situation where NK cells come into contact with non-self. The ILT family consists of at least eight members with different numbers of Ig domains and again there are activating and inhibitory forms. However, unlike KIR, whose expression is almost entirely restricted to NK cells, ILT are found predominantly on cells of the monocytic lineage, B cells and also on NK and T cell subsets. Only two members of the ILT family (ILT2 and ILT4) have so far been found to bind to a broad range of HLA class I molecules including HLA-G (Long, 1999).
King et al.: Trophoblast HLAI Molecules Recognition
UTERINE NK CELL RECEPTORS FOR TROPHOBLAST HLA CLASS I MOLECULES The NK cells in the uterine mucosa are a distinctive cell type (CD56bright, CD16) whose relationship with circulating CD56 + NK cells is unknown (King et al., 1998). Once it became obvious that NK cells as well as T cells were capable of allorecognition, the possibility that maternal uterine NK cells used this allorecognition system to sense the presence of the semi-allogeneic fetus was raised (King and Loke, 1991). The close association between these unusual NK cells and EVT also suggested the existence of NK cell receptors which could potentially recognize trophoblast HLA-G, HLA-C and HLA-E. This NK cell/trophoblast interaction could result in modulation of the invasiveness of the EVT and, therefore, the extent of vascular transformation and the maternal blood flow to the fetus.
HLA-E Virtually all decidual CD56bright decidual NK cells express CD94/NKG2A at five times a greater intensity than blood NK cells as measured by flow cytometry. The coexpression of CD94/NKG2C cannot be determined as there are no Mabs available to the NKG2C molecule. However, NKG2C transcripts are present in FACS-sorted CD56bright cells by RT-PCR (unpublished). We have recently demonstrated that more than 95 per cent of decidual NK cells will bind to soluble HLA-E tetrameric complexes and that this binding is inhibited by Mabs to CD94 (King et al., submitted). Therefore, HLA-E could act as a sentinel molecule sensitive to levels of expression of other HLA class I molecules. In addition, the particularly high affinity for HLA-E molecules with the HLA-G-derived leader sequence bound in its groove means that maternal uterine NK cells can potentially sense and respond to the HLA-G + fetal trophoblast.
HLA-C KIR2D molecules specific for HLA-C are expressed by decidual NK cells (Verma et al., 1997; Hiby et al., 1997). These KIR have been demonstrated by RT-PCR and sequencing of CD56bright cells sorted to a high degree of purity by flow cytometry, by immunostaining of tissue sections and by flow cytometric analysis of dissociated decidual leukocytes. No unique KIR sequences were found which had not been isolated from blood NK cells, so the potential repertoire used by maternal uterine NK cells is the same as for blood NK cells. However, using Mabs GL183 and EB6, we found that a higher proportion of CD56bright NK cells were GL183 + and/or EB6 + compared to peripheral blood NK cells analysed at the same time from a pregnant individual. (Mabs GL183 and EB6 are specific for KIR2D receptors which bind to the two groups of HLA-C allotypes). At 8–10 weeks gestation, 50–80 per cent of
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decidual NK cells were reactive with either or both Mabs compared to only 5–20 per cent in peripheral blood CD56 + cells (Verma et al., 1997). Therefore, the maternal uterine NK cells appear skewed towards HLA-C recognition compared to those in peripheral blood.
HLA-G A receptor expressed by decidual NK cells which is specific for HLA-G has remained intriguingly elusive. Early reports that members of the KIR family specific for HLA-C or HLA-B could also bind HLA-G were not subsequently confirmed. It was then thought that CD94/NKG2 mediated HLA-G recognition, but these data are now explained by the coexpression of HLA-E on the target cells used in the cytotoxicity assays (Long, 1999). Two other receptors are now candidates for the HLA-G receptor. Members of the ILT2 family were found to bind to a wide range of HLA class I molecules, including HLA-G in cytotoxicity assays, and by direct binding of soluble ILT fusion proteins to cells transfected with single HLA class I molecules (Long, 1999). When soluble tetrameric complexes of HLA-G were tested for binding to peripheral blood mononuclear cells it was found that only a subset of blood monocytes were stained and there was no binding at all to NK cells. Binding of the HLA-G tetramers was found to be mediated by ILT receptors, mainly by ILT4 with some contribution by ILT2, but only when the latter was expressed at high levels (Allan et al., 1999). ILT2 is expressed by 20–25 per cent of decidual NK cells in all women and both ILT2 and ILT4 are present on all decidual macrophages (Verma et al., 1997 and unpublished). However, the broad specificity of ILT2 and ILT4 for many HLA class I molecules would suggest that HLA-G is not providing a unique ‘fetal’ signal to maternal leukocytes. These results suggest that HLA-G could modulate maternal macrophage, rather than NK cell, behaviour at the maternal– fetal interface. Recently, there is evidence that one of the orphan (i.e. with no recognizable ligand) KIR, KIR2DL4, will bind to HLA-G (Lanier, 1999; Rajagopalan and Long, 1999; Ponte et al., 1999) (Figure 3). When expressed at high levels in an NK cell line, KIR2DL4 inhibited killing of a target cell transfected with HLA-G. In addition, soluble KIR2DL4 fusion proteins bound to HLA-G transfectants, but not to similar cells transfected with other HLA class I molecules. KIR2DL4 is different from other KIR in expression and structure. Amongst its unique structural features is the combination of a cytoplasmic domain containing a single ITIM with a charged residue in the transmembrane region. This suggests it might be ‘schizophrenic’ in function, having both inhibitory ITIM functions and also the ability to associate with another activating molecule. The expression pattern is also of interest, as all other KIR are expressed on overlapping NK cell subsets. Whilst one report has described expression on all NK cells (Rajagopalan and Long, 1999), another has only found a subset of decidual
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NK cells from early pregnancy are KIR2DL4 + whilst all blood NK cells are negative (Ponte et al., 1999). Development of more reagents should resolve this issue and lead to investigation of the functional consequences of KIR2DL4 interaction with HLA-G in the decidua basalis. The challenge now is to reconcile all these observations. It is clearly important to establish whether HLA-G is predominantly a target recognition molecule for either decidual NK cells and/or macrophages. The binding assay using the HLA-G tetrameric complexes demonstrated no staining of peripheral blood NK cells. In contrast, there was binding of KIR2DL4 fusion proteins to HLA-G transfectants. The possible explanations to explain these conflicting results are as follows: (1) Although HLA-G tetramers did bind to ILT2 transfectants this only occurred if ILT2 was expressed at high levels. The expression levels of ILT2 normally on NK cells may be too low to show detectable binding with the HLA-G tetramers, but this interaction could still be functionally important; (2) The HLA-G tetramers were refolded in vitro around a self-peptide. Whilst KIR recognition is dependent on the sequence at positions 7 and 8 of the bound peptide, ILT recognition is independent of the peptide sequence. Perhaps refolding of the HLA-G protein around a variety of peptides found from elution studies to bind to HLA-G would give different results; (3) The HLA-G protein used to form the tetrameric complexes was prepared in Escherichia coli and would, therefore, not be glycosylated. HLA-G is much more heavily glycosylated when expressed by normal trophoblast compared to JEG-3 choriocarcinoma cells or HLA-G transfectants (McMaster and Fisher, 1998). There is one glycosylation site on HLA class I molecules which is at position 86 on the 1 domain close to the KIR recognition site (77–83). There is still no evidence that glycosylation influences human NK cell recognition despite the suspicions raised by the possession of a carbohydaterecognition domain on the C-type lectin NK cell receptors (Braud et al., 1998). However, MHC-linked carbohydrates may influence NK cell binding in the mouse.
FUNCTIONAL IMPLICATIONS The discovery of NK cell receptors for HLA class I molecules has depended heavily on cytotoxicity assays using target cells transfected with various class I molecules. The use of phenotypically well-defined peripheral blood NK cell clones expressing individual NK cell receptors and specific Mabs has allowed dissection of individual NK cell receptor-HLA class I ligand interactions which either inhibit or activate the NK cell. Direct binding assays using soluble fusion proteins of KIR, ILT and CD94/NKG2 molecules have also been used and the recent development of soluble HLA tetramers has been a
Placenta (2000), Vol. 21, Supplement A, Trophoblast Research, Vol. 14
significant technical advance. There is still difficulty, however, in extrapolating these in vitro assays to the in vivo situation at the implantation site. For example, there are practical and ethical difficulties in generating decidual NK cell clones. Unusual configurations and the glycosylation of HLA class I molecules on trophoblast also means that B cell transfectants are not ideal targets. We have used polyclonal decidual NK cells isolated separately from different individuals to see what the overall effect of recognition of different HLA class I molecules is on cytotoxicity. The high levels of CD94/NKG2A expression do mediate inhibition of killing of decidual NK cells when exposed to target cells expressing HLA-E, and this inhibition is partially reversed in the presence of anti-CD94 Mabs (King et al., submitted). Thus, even if there does turn out to be coexpression of CD94/NKG2C on all the cells, the dominant effect on killing is inhibitory. Trophoblasts may not be the only cells in the decidua basalis which need to survive. NK cells are not generally a tissue cell and are found mainly in blood and, to a lesser extent, in the spleen and bone marrow. There must be fail-safe mechanisms to prevent lysis of the surrounding autologous maternal cells and this may be why CD94/NKG2 is upregulated in the decidua. Therefore, HLA-E could be the survival molecule for all these tissue cells. There is also functional evidence that killing by NK clones derived from peripheral blood or polyclonal decidual NK cells is inhibited by HLA-G. This interaction appears dependent on HLA-G rather than HLA-E, because Mabs specific for HLA-G which have no cross-reactivity to HLA-E partially reverse the inhibition (Navarro, 1999; King et al., submitted). Decidual NK cells will also be able to sense the HLA-G expression by trophoblast through the presentation of the HLA-G leader sequence by HLA-E, and by the expression of ILT2 on a minor subset which binds to HLA-G at low affinity. Because HLA-G is monomorphic it could be viewed as an invariant ‘fetal’ signal signifying that pregnancy has occurred. In contrast, the addition of the anti-KIR Mabs, GL183 and EB6, to cytotoxicity assays using HLA-C transfectants has no effect on killing, despite the high proportion of cells staining with these Mabs. As with CD94/NKG2, we are still unsure whether these KIR are predominantly inhibitory forms or whether activating forms are also abundant. EB6 and GL183 cannot distinguish between these forms but both are transcribed (Hiby et al., 1997). The interest of HLA-C lies in its polymorphism which leads to a potentially different fetal HLA-C from the mother’s HLA-C, and varies in different pregnancies. There is also variability on the maternal side as the KIR genotype differs between individuals. Therefore, the outcome of each pregnancy is different, depending on the maternal NK cell genotype and the paternal HLA-C allotype expressed in the fetus. Because of our original hypothesis that trophoblast survival depended on the expression of the non-classical HLA-G molecule (King and Loke, 1991), we have also performed these cytotoxicity assays using normal trophoblast as target cells in the presence of Mabs to HLA class I molecules, HLA-G,
King et al.: Trophoblast HLAI Molecules Recognition
HLA-E
CD94/NKG2
HLA-C
KIR
HLA-G
ILT4 (+ILT2) KIR2DL4
Figure 4. Decidual NK cell recognition of trophoblast HLA class I molecules.
CD94/NKG2, ILT2 and KIR. We have not been able to detect any lysis of trophoblast, in keeping with another report using JEG-3 cells and blood NK cells (King et al., submitted; Avril et al., 1999). This raises the question of what other effector functions may be triggered by NK cell recognition of trophoblast. The role of NK cells in providing defence against viral infection is widely known but, interestingly, in vivo there is only firm evidence that this is mediated by NK cell cytokines and not by cytotoxicity (Biron et al., 1999). Similarly, in hybrid resistance in mice, an experimental system of NK cell-mediated allorecognition, it is failure of engraftment of the donor cells due to lack of beneficial NK cell cytokines which leads to apparent ‘rejection’ (Aguila and Weissman, 1996). Thus, the focus now should probably be on the cytokines produced by uterine NK cells in response to binding to trophoblast HLA class I molecules. Despite the lack of evidence to show how NK cells influence EVT behaviour, these recent results have, for the first time, provided molecular mechanisms to explain how the mother might recognize the semi-allogeneic fetus (Figure 4).
REFERENCES Aguila HL & Weissman IL (1996) Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells. Blood, 87, 1225–1231. Allan DSJ, Colonna M, Lanier LL, Churakova TD, Abrams JS, Ellis SA, McMichael AJ & Braud VM (1999) Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells. J Exp Med, 189, 1149–1155. Avril T, Jarousseau AC, Watier H, Boucrat J, Le Bouteiller P, Bardos P & Thibault G (1999) Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism. J Immunol, 162, 5902– 5909. Biron CA, Nguyen KB, Pien GC, Cousens LP & Salazar-Mather TP (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines. Ann Rev Immunol, 17, 189–220. Braud VM, Jones EY & McMichael A (1997) The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol, 27, 1164–1169.
S85 Braud VM, Allan DSJ, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, Lanier LL & McMichael AJ (1998) HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391, 795–799. Hiby SE, King A, Sharkey AM & Loke YW (1997) Human uterine NK cells have a similar repertoire of Killer Inhibitory and Activatory receptors to those found in blood, as demonstrated by RT-PCR and sequencing. Mol Immunol, 34, 419–430. Hiby SE, King A, Sharkey A & Loke YW (1999) Molecular studies of trophoblast HLA-G: polymorphism, isoforms, imprinting and expression in pre-implantation embryo. Tissue Antigens, 53, 1–13. King A & Loke YW (1991) On the nature and function of human uterine granular lymphocytes. Immunol Today, 12, 432–435. King A, Burrows TD, Hiby SE, Joseph S, Verma S, Lim PB, Gardner L, Bowen JM, Ziegler A, Uchanska-Ziegler B, Le Bouteiller P & Loke YW. (2000) Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta, in press. King A, Allan DSJ, Joseph S, Verma S, Hiby SE, Bowen JM, McMichael AJ, Braud VM & Loke YW. HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Submitted. King A, Boocock C, Sharkey A, Gardner L, Beretta A, Siccardi AG & Loke YW (1996) Evidence for the expression of HLA-C class I mRNA and protein by human first trimester trophoblast. J Immunol, 156, 2068–2076. King A, Burrows T, Verma S, Hiby SE & Loke YW (1998) Human uterine lymphocytes. Hum Reprod Update, 4, 480–485. Lanier LL (1998) NK cell receptors. Ann Rev Immunol, 16, 359–393. Lanier LL (1999) Natural killer cells fertile with receptors for HLA-G? Proc Nat Acad Sci USA, 96, 5343–5345. Ljunggren H-G & Ka¨rre K (1990) In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today, 11, 237–244. Loke YW & King A (1995) Human Implantation: Cell Biology and Immunology. Cambridge: Cambridge University Press. Long EO (1999) Regulation of immune responses through inhibitory receptors. Ann Rev Immunol, 17, 875–904. McMaster MT & Fisher SJ (1998) The fetal transplant: is HLA-G important? In Fertility and Reproductive Medicine (Eds) Kempers RD, Cohen J, Haney AF & Younger JB, pp. 661–678. Amsterdam: Elsevier Science B.V. Navarro F, Llano M, Bellon T, Colonna M, Geraghty DE & LopezBotet M (1999) The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognise HLA-G1 and HLA-E molecules co-expressed on target cells. Eur J Immunol, 29, 277–283. Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G, Vitale C, Bertone S, Moretta A, Moretta L & Mingari MC (1999) Inhibitory receptors sensing HLA-G1 molecules in pregnancy: deciduaassociated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proc Nat Acad Sci USA, 96, 5674–5679. Rajagopalan S & Long EO (1997) The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)Cw4 exhibits peptide selectivity. J Exp Med, 185, 1523–1528. Rajagopalan S & Long EO (1999) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J Exp Med, 189, 1093–1100. Vale´s-Go´mez M, Reyburn HT, Erskine RA, Lo´pez-Botet M & Strominger JL (1999) Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. EMBO J, 18, 4250–4260. Verma S, King A & Loke YW (1997) Expression of killer-cell inhibitory receptors (KIR) on human uterine NK cells. Eur J Immunol, 27, 979–983.