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. 2011 Apr 5;17(9):577–586. doi: 10.1093/molehr/gar022

Ex vivo functional responses to HLA-G differ between blood and decidual NK cells

Richard Apps 1,2, Andrew Sharkey 1, Lucy Gardner 1, Victoria Male 1, Pippa Kennedy 1, Leanne Masters 1, Lydia Farrell 1, Des Jones 1, Rasmi Thomas 2, Ashley Moffett 1,*
PMCID: PMC3160205  PMID: 21471023

Abstract

Restricted expression of human leucocyte antigen-G (HLA-G) to fetal extravillous trophoblast cells, which invade the decidua during implantation, suggests a role for HLA-G in placentation. In this study, we have investigated several aspects of HLA-G expression and function. Surface levels of HLA-G expression were measured in 70 normal pregnancies. We show the dimeric conformation that is unique to HLA-G forms after passage through the Golgi apparatus. Differences were found in the receptor repertoire of decidual natural killer (dNK) cells that express the leucocyte immunoglobulin-like receptor B1 (LILRB1), which binds dimeric HLA-G strongly. We then measured functional responses of dNK cells with LILRB1, when stimulated by HLA-G in both monomeric and dimeric conformations. Degranulation, interferon-γ and interleukin-8 production by dNK cells freshly isolated from the first trimester implantation site were either undetected or not affected by HLA-G. These findings should be considered when inferring the activity of tissue NK cells from results obtained with cell lines, peripheral NK or cultured dNK cells.

Keywords: HLA-G, immunology, implantation, leucocytes, trophoblast

Introduction

Human leucocyte antigen-G (HLA-G) is a non-classical HLA class I molecule with expression restricted to extravillous trophoblast (EVT) cells of the fetal placenta (Kovats et al., 1990; Apps et al., 2008). Villous trophoblast cells express no HLA molecules, but as cells move away from the placental villus in cytotrophoblast cell columns, HLA-G expression is switched on and all EVT populations invading the decidua and spiral arteries show strong HLA-G expression (Moffett and Loke, 2006; Apps et al., 2009). EVTs are thus the first allogeneic fetal cells to contact maternal uterine immune cells, and this pattern of HLA-G expression suggests a role in signalling to decidual leucocytes. EVT invasion is important to the success of a human pregnancy, as it transforms decidual arteries to increase blood supply to the placenta, and decidual leucocytes are thought to regulate this invasion (Brosens, 1977; Khong et al., 1986).

The majority of decidual leucocytes are CD56bright natural killer (NK) cells that are distinct from peripheral blood NK (PBNK) cells (Moffett-King, 2002; Koopman et al., 2003). Functional effects of HLA-G binding to NK cells have been clearly shown with PBNK. HLA-G transfected into class I-negative target cells inhibits cytotoxicity and interferon (IFN)-γ production by PBNK cells and NK clones (Perez-Villar et al., 1997; Morel and Bellon, 2008). This inhibition occurs by two pathways: stimulation of the leucocyte immunoglobulin-like receptor B1 (LILRB1; ILT2, LIR-1, CD85j) directly and by HLA-G providing a peptide derived from the HLA-G leader sequence that induces surface expression of the CD94/NKG2A ligand, HLA-E (Colonna et al., 1997; Navarro et al., 1999). These assays show responses to HLA-G that are the same as for classical HLA class I molecules interacting with PBNK.

More recently, it has emerged that HLA-G may also act differently to other class I molecules because two conventional β2m-associated complexes of HLA-G form a homodimer via a disulphide bond at position 42 in the α2 domain (Boyson et al., 2002). This dimeric conformation is unique to HLA-G among HLA class I molecules and substantially increases LILRB1 binding, measured by binding of Fc-fusion proteins in flow cytometry or recombinant soluble molecules in surface plasmon resonance (Gonen-Gross et al., 2003; Shiroishi et al., 2006). Increased binding avidity of the HLA-G dimer translates into augmented LILRB1 signalling, shown by inhibition of IgɛR-mediated serotonin release, NK cytotoxicity and a chimeric LILRB1 reporter cell assay (Gonen-Gross et al., 2003; Shiroishi et al., 2006). Most recently, LILRB1 has been shown to bind HLA-G expressed on trophoblast cells in vivo, with the HLA-G homodimer being preferentially immunoprecipitated from the cell surface by LILRB1-Fc fusion protein (Apps et al., 2007). LILRB1 is expressed by all HLA-DR+ myelomonocytic cells and by a subset of CD56bright cells in the decidua (Apps et al., 2007; El Costa et al., 2008), similar to its expression in peripheral leucocytes (Colonna et al., 1997; Fanger et al., 1998; Morel and Bellon, 2008; Yawata et al., 2008).

We here characterize HLA-G expression, dimer formation and LILRB1+ NK cells at the first trimester implantation site. We then investigate the outcome of HLA-G-stimulating LILRB1 on NK cells. Degranulation, IFN-γ and interleukin (IL)-8 production by decidual NK (dNK) cells isolated from normal first trimester pregnancies are measured. dNK responses to stimulation by target cells expressing different conformations of HLA-G are investigated.

Materials and Methods

Primary tissue, cell lines and co-cultures

Decidual and placental tissue was obtained from elective terminations of normal pregnancies between 6 and 12 weeks of gestation. Ethical approval for the use of these tissues was obtained from the Cambridge Local Research Ethics Committee. Samples of decidua were frozen in liquid nitrogen for histological analysis. Trophoblast and leucocytes were isolated as described previously (Male et al., 2010a). Briefly, trophoblast was released from chorionic villi by trypsin digestion, and after overnight culture on fibronectin, 50–80% of the cells express HLA-G, a marker unique to EVT cells (Apps et al., 2008). Leucocytes were isolated by collagenase digestion of maternal decidual tissue and either stained or cultured with target cells immediately or cultured in RPMI with 10% fetal calf serum (FCS) and 5 ng/ml of rIL-15 (Peprotech) for up to 3 days before degranulation assays. Where added, 4β-phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) was at 160 nM. Peripheral blood leucocytes were isolated on Lymphoprep (Axis-Shield) from fresh venous blood of normal adult volunteers. Cell lines used were the HLA-I-null human B lymphoblastoid 721.221 line (Shimizu et al., 1988) and 721.221 transfected with HLA-G (Navarro et al., 1999) or a C42S HLA-G mutant unable to dimerize (Gonen-Gross et al., 2003). For degranulation assays, 2 × 105 target 721.221 cells were plated with 5 × 105 peripheral or 2.5 × 105 decidual leucocytes in a total volume of 150 μl medium in U-bottomed 96-well plates and centrifuged (100g for 3 min) before culture for 4 h.

Flow cytometry

Monoclonal antibodies (mAbs) used in this study to bind HLA-G were G233 (Loke et al., 1997)—made in our own laboratory—and MEM-G/9-FITC (Menier et al., 2003)—purchased from Serotec. Tu149 (Uchanska-Ziegler et al., 1993), which is specific to HLA-C on EVT cells (Apps et al., 2009), was kindly supplied by B. Uchanska-Ziegler. GHI/75, which binds LILRB1 (Banham et al., 1999), and ZM3.8, which binds LILRB3 (Cella et al., 1997), were purchased conjugated to PE-Cy5 from Becton Dickinson and Beckman Coulter. EB6 (KIR2DL1/S1; Moretta et al., 1993), GL183 (KIR2DL2/L3/S2; Moretta et al., 1993), DX9 (KIR3DL1; Litwin et al., 1994) and Z199 (NKG2A; Perez-Villar et al., 1996) were purchased unconjugated or conjugated to PE from Beckman Coulter. CD56-FITC, CD56-PE and CD56-PC5 (clones B159, MY31 and N901), CD107a-FITC (clone H4A3), HLA-DR-FITC (clone L243) and isotype controls X39 and X40 were obtained from Beckman Coulter and Becton Dickinson for surface staining. IFN-γ-FITC (clone 25723.11), IL-8-PE (clone AS14) and isotype control preparations for intracellular staining of MOPC-21-PE and 27–35-FITC were purchased from BD Pharmingen and R&D Systems. Binding of unlabelled mAbs was detected by polyclonal FITC- or PE-conjugated secondary antibody to murine immunoglobulin G (IgG; both Sigma-Aldrich).

Freshly isolated cells or those harvested from culture, adherent trophoblast being dissociated with trypsin (Becton Dickinson), were resuspended in FACS buffer [1% FCS, 1 mM EDTA in phosphate-buffered saline (PBS)] and incubated with human IgG (Sigma-Aldrich) before unlabelled primary mAbs then fluorochrome-conjugated polyclonal secondaries. Free secondary antibody-binding sites were blocked with murine immunoglobulin (Sigma-Aldrich) before staining with directly conjugated mAb to identify leucocyte or trophoblast cell populations. For intracellular staining, cells were first stained for surface antigens as mentioned already, fixed for 15 min in 3% paraformaldehyde, permeabilized by washing in FACS buffer with 0.1% saponin (Sigma-Aldrich) and then incubated with conjugated mAb to intracellular cytokine. When using cultured cells, 4 h prior to staining, brefeldin A (Sigma-Aldrich) was added to the culture medium to a concentration of 10 µg/ml. To measure NK cell degranulation, monensin GolgiStop (BD Biosciences) and CD107a-FITC mAb were added to the culture medium 4 h prior to harvesting and staining for other surface antigens. Cells were analysed using a FACscan flow cytometer and CellQuest software (Becton Dickinson).

Genotyping

For fetal samples from which trophoblast cells were prepared, genomic DNA was isolated from macroscopically identified chorionic villi. Digestion with proteinase K and RNase A (both Roche) in the presence of tissue lysis then protein precipitation buffer (both Qiagen) was performed according to the manufacturer's instructions. DNA was then pelleted with isopropanol and resuspended in Tris 10 mM and EDTA 0.1 mM, pH 7.5. The 14 bp insertion/deletion polymorphism in the 3′-untranslated region (UTR) of HLA-G was typed as in Harrison et al. (1993), by amplifying a 141 or 155 bp region spanning the polymorphism using the primers 5′-GTAGTGTGAAACAGCTGCCC-3′ and 5′-AAGGAATGCAGTTCAGCATGA-3′ and resolving products by electrophoresis with a 3% low-melting-point agarose gel. The single-nucleotide polymorphism in the 3′-UTR of HLA-G, at the miR-148a/b-binding site, was typed by PCR with the primers 5′-TCTCCTGCAACAAATCAGCAC-3′ and 5′-AAGGGGCTGGGATGTC-TCCG-3′ and sequencing of products using the same primers and an ABI 3730 DNA analyzer (Applied Biosystems).

Pulse chase and immunoprecipitation

721.221 cells transfected with HLA-G were starved for 30 min in the medium lacking cysteine and methionine, labelled for 20 min with 0.1 mCi/ml l-[35S]methionine and l-[35S]cysteine (Promix, GE Healthcare) and chased in a regular medium for the indicated times. Cells were lysed and immunoprecipitated as described previously (Apps et al., 2007). Digestion with endoglycosidase Hf was performed according to the manufacturer's instructions (New England Biolabs). Samples were resolved on non-reducing NuPAGE Bis-Tris 10% gels (Invitrogen), dried and exposed to autoradiography film (GE Healthcare).

Immunohistology and immunofluorescence

Immunohistology was performed as described previously (Kam et al., 1999). Briefly, frozen sections of decidua were acetone-fixed and rehydrated. Staining with the anti-HLA-G mAb G233 was detected using Vectastain elite ABC alkaline phosphatase kit (Vector Laboratories) and FAST RED substrate (Sigma-Aldrich) before counterstaining with Carazzi's haematoxylin. For immunofluorescence, serial sections were blocked with 2% goat serum and 1% bovine serum albumin in PBS. Anti-CD56-FITC (Serotec, clone MEM-188) and anti-LILRB1 (Amgen, clone M401) in PBS/2% goat serum were applied for 30 min. M401 was detected using anti-mouse-IgG1-AlexaFluor568, and CD56-FITC was amplified using anti-mouse IgG2a-AlexaFluor488 (both Invitrogen) for 60 min. Cover slips were mounted with Vectashield-DAPI (Vector laboratories) and images collected using an Axiophot fluorescence microscope (Zeiss). For each implantation site, the number of single CD56+, single LILRB1+ and double CD56/LILRB1+ cells was counted in 20 viewfields which included areas both invaded and uninvaded by EVT.

Results

Characterization of HLA-G on EVT

Although levels of HLA-G mRNA have been found to correlate with two variants in the 3′-UTR [a 14 bp insertion/deletion polymorphism (Rousseau et al., 2003) and a separate SNP in the binding site of microRNAs 148a and b (Tan et al., 2007)], no studies have investigated variation in the level of HLA-G surface protein expressed in vivo. To do this, we measured the levels of HLA-G on EVT cells isolated from 70 normal first trimester pregnancies by flow cytometry. We found little variability in the level of HLA-G expression by EVT cells from different donors, certainly less than that of HLA-C (Fig. 1). The limited variation in HLA-G surface protein expression that was observed showed no correlation with either the 14 bp insertion/deletion, or miR-148a/b-binding site polymorphisms. Our results from primary cells suggest that HLA-G is a consistent signal in pregnancy.

Figure 1.

Figure 1

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Variation in the level of HLA-G expression between placental samples. (A) Preparations of placental cells from normal first trimester pregnancies were gated on scatter (R1) and ILT3 + macrophages excluded (R2). HLA-G expression on EVT cells was measured by flow cytometry using the mAb MEM-G/9. HLA-C expression on HLA-G + EVT was measured by staining with mAb Tu149 (open trace) or isotype control (filled). (B) Median fluorescence intensity of antibodies binding HLA-G and HLA-C on EVT is shown for 70 independent donors. Open points show staining with isotype control mAb. (C) No correlation with HLA-G surface protein expression was observed for either the 14 bp insertion/deletion or the miR-148a/b-binding site polymorphisms in the 3′-UTR of HLA-G.

HLA-G is unusual not only because of its restricted tissue expression to extra-embryonic cells but also because it is the only class I molecule that forms a β2m-associated homodimer at the cell surface. As well as normal trophoblast, the dimeric form of HLA-G has also been demonstrated on transfected cells. Metabolic labelling followed after increasing chase by immunoprecipitation of HLA-G from transfected 721.221 cells shows that the dimeric form of HLA-G is completely endoH-resistant and does not appear until significant amounts of the HLA-G monomer become endoH-sensitive (Fig. 2). This is consistent with the disulphide bond of the HLA-G dimer forming after passage through the Golgi apparatus, most likely at the cell surface.

Figure 2.

Figure 2

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Formation of the dimeric conformation of HLA-G. HLA-G-transfected 721.221 cells were labelled with 35S for 25 min and immunoprecipitated with the mAb G233 after the indicated chase times (hours). Precipitated antigens were incubated with or without endoglycosidase H digestion before resolution by non-reducing SDS–polyacrylamide gel electrophoresis. The dimeric form of HLA-G is always endoH-resistant and does not appear until a significant amount of monomeric HLA-G has exited the Golgi apparatus.

Characterization of dNK cells expressing LILRB1

The major ligand of the HLA-G dimer is LILRB1, which is expressed by a subset (20–50%) of dNK cells. Given the variegated and overlapping expression of NK receptors for major histocompatibility complex (MHC), we compared by flow cytometry the repertoire of receptors recognizing HLA class I molecules between dNK cells with and without LILRB1. The frequencies of NKG2A and KIR3DL1 were the same on LILRB1+/− dNK cells, but killer immunoglobulin-like receptor (KIR) that bind Group 2 HLA-C molecules were significantly (P < 0.05) more likely to be expressed by dNK cells that also express LILRB1 (Fig. 3A–E). To see the anatomical location of LILRB1+ and LILRB1− dNK in the decidua, we performed immunohistology. NK cells in the decidua basalis (the site of trophoblast invasion) and decidua parietalis (decidua away form the site of placentation) expressed similar frequencies of LILRB1 (Fig. 3F–H).

Figure 3.

Figure 3

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Distribution and receptor repertoire of LILRB1+/− dNK cells. Decidual leucocytes were isolated from normal first trimester pregnancies and analysed by flow cytometry. NK cells were identified by scatter and CD56 labelling, and then stained for LILRB1 expression (A–C). Representative staining of KIR and NKG2 receptors on LILRB1− (grey) or LILRB1+ (black) NK cells is shown (D). The proportion of cells expressing these receptors is shown from seven independent donors. KIR2DL1/S1 is expressed significantly more frequently on LILRB1+ dNK cells (P = 0.05 by two-tailed Wilcoxon signed-rank test) (E). The localization of LILRB1+/− dNK cells in vivo was then investigated. A representative implantation site is shown stained for HLA-G by light microscopy to identify regions of EVT invasion (F), and an example of double labelling for CD56 and LILRB1 by immunofluorescence in these sections is shown (G). The proportion of NK cells expressing LILRB1 in regions of decidua invaded and uninvaded by EVT is then summarized from four independent individuals (H).

Effect of HLA-G on dNK cell degranulation

dNK cells are reported to produce the pro-inflammatory cytokines IFN-γ and IL-8 but when we stained freshly isolated preparations of decidual leucocytes by intracellular flow cytometry, only very small amounts were detected in CD56+ NK cells irrespective of whether stained immediately (Fig. 4A and B) or after 4–6 h culture with brefeldin A (data not shown). IL-8 production was primarily seen in HLA-DR+ cells, which are predominantly macrophages in our decidual leucocyte preparations (Gardner and Moffett, 2003). IFN-γ was produced after 6 h stimulation with PMA, confirming that dNK cells can produce this cytokine. Given these indications that the reported findings of IFN-γ and IL-8 production by dNK cells are in response to in vitro stimulation, we tested dNK cell degranulation when cultured with HLA class I-null target cells (Fig. 4C). An average of around 5% of freshly isolated dNK cells become CD107a+ in response to 721.221 target cells, which increases to around 20% when using dNK first stimulated with 5 ng/ml IL-15 for 3 days (Fig. 4D).

Figure 4.

Figure 4

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IFN-γ, IL-8 and CD107 as read-outs of freshly isolated decidual leucocyte function. Decidual leucocytes from normal first trimester pregnancies were analysed immediately after isolation by intracellular flow cytometry for the production of IFN-γ (A) or IL-8 (B). Degranulation was assessed after 4 h of culture with HLA class I-negative target cells (C). Decidual leucocytes were gated by scatter and identified by labelling of CD56 or HLA-DR. Compared with an isotype control, little staining for IFN-γ is observed without PMA stimulation (A). The anti-IL-8 mAb stained some freshly isolated decidual leucocytes, but these were HLA-DR+ not CD56+ cells (B). Representative staining for surface CD107a expression by CD56+ NK cells cultured alone and with HLA class I-null 721.221 target cells is shown for dNK cells freshly isolated or first stimulated with IL-15 for 3 days (C). The proportion of CD56+ cells that become CD107a+ after culture with 721.221 targets is then shown for 20 independent donors (D). CD107 up-regulation is quantified by subtracting the CD107 staining detected on NK cells cultured without target cells from that of cultures with target cells.

We then sought to investigate modulation by HLA-G of dNK cell degranulation responses to target cells. NK cell degranulation by IL-15-stimulated dNK was compared in response to 721.221 transfectants expressing HLA-G (Fig. 5). Neither monomeric nor dimeric forms of HLA-G affected degranulation of dNK cells (Fig. 5D). This was still the case when the LILRB1+ subset of dNK cells was specifically identified by flow cytometry (Fig. 5E). Degranulation of PBNK cells was inhibited by HLA-G, consistent with previous reports (Perez-Villar et al., 1997; Morel and Bellon, 2008). Therefore, although these results confirm the effect of HLA-G in inhibiting cytotoxicity of PBNK, similar functional read-outs were not affected by the interaction of dNK cells with HLA-G.

Figure 5.

Figure 5

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dNK cell degranulation is not inhibited by target cells expressing different conformations of HLA-G. 721.221 cells transfected with HLA-G (green) or a C42S mutant unable to dimerize (red) were sorted for identical expression levels and compared with untransfected 721.221 cells (grey), monitored by mAb MEM-G/9 staining (A). Lymphocyte preparations were cultured for 3 days with IL-15 and then degranulation of NK cells was detected using flow cytometry for CD107a expression in response to 721.221 target cells expressing HLA-G in monomeric or dimeric conformations. Representative staining is shown of peripheral leucocytes (B) or decidual leucocytes from normal first trimester pregnancies (C). Modulation of NK cell degranulation by the presence of HLA-G is shown for 17 independent donors (D). When the LILRB1+ dNK cells are specifically identified, there is still no evidence for either form of HLA-G-inhibiting degranulation (E).

Discussion

Our results characterize the signal potentially delivered by HLA-G on trophoblast cells to local uterine leucocytes. Although HLA-G has limited polymorphism, differences in the levels of HLA-G between individuals have been reported to be associated with particular variants (Hviid et al., 2003; Rousseau et al., 2003; Tan et al., 2007; Larsen and Hviid, 2009). These studies either analysed protein expression in transfected cells or measured only mRNA levels. We have analysed HLA-G surface protein expression in vivo by flow cytometry on EVT cells isolated from 70 normal first trimester pregnancies. The limited variation in the level of HLA-G that was observed did not correlate with variants previously shown to influence expression in vitro—either a 14 bp insertion/deletion (Rousseau et al., 2003) or miRNA-148a/b-binding site polymorphisms in the 3′-UTR of HLA-G (Tan et al., 2007). Variation in HLA-G levels was also clearly less than that observed for HLA-C surface expression on the same samples. Restricted expression of HLA-G to invasive trophoblast cells, as well as consistent level of surface expression, suggests that HLA-G gives a similar pregnancy-specific signal to uterine leucocytes in every gestation.

It has been difficult to establish the function of HLA-G during placentation. dNK cell expression of KIR2DL4 and binding of this receptor to HLA-G has not been consistently replicated (Apps et al., 2008). The strongest evidence for a decidual leucocyte receptor interacting with HLA-G is currently that for LILRB1 (Apps et al., 2008). LILRB1 binds all HLA class I molecules but HLA-G uniquely exists in a dimeric conformation that considerably increases LILRB1 binding (Boyson et al., 2002; Gonen-Gross et al., 2003; Shiroishi et al., 2006; Apps et al., 2007). We performed metabolic labelling and pulse-chase experiments to show that the disulphide bond formation of the HLA-G dimer does not occur until after passage through the Golgi apparatus, providing further evidence that the dimers are not a post-lysis artefact. How the disulphide bonds form is not clear, but this may involve surface disulphide isomerize activity recently shown to be important in modification of MHC class I-related molecules (Kaiser et al., 2007).

The receptor repertoire and distribution at the implantation site of dNK cells with and without LILRB1 was compared within individuals. NKG2A expression and anatomical distribution were the same for both LILRB1+/− NK cells. KIRs were expressed at a higher frequency by dNK cells also expressing LILRB1, a trend reaching statistical significance for KIR2DL1/S1. As immature NK cells have been found in the uterine mucosa (Male et al., 2010b), and KIR/LILR are known to be acquired late during NK cell maturation (Bjorkstrom et al., 2010; Lopez-Verges et al., 2010), LILRB1+ NK cells likely represent a late developmental stage in the decidua. To investigate the functional effects of HLA-G on dNK cells, we initially looked for modulation of established assays of NK activity. Only low levels of IFN-γ staining were detected by intracellular flow cytometry of dNK cells analysed immediately after isolation. This is consistent with previous intracellular cytometry experiments using fresh decidual (Sharkey et al., 2008) or endometrial NK cells (Eriksson et al., 2006), although does contrast with an apparent role of IFN-γ in murine pregnancy (Ashkar and Croy, 2001). IL-8 is a reported dNK product, but our staining shows that macrophages are the major source of this cytokine in freshly isolated decidual leucocytes. A previous study detected strong IL-8 staining of dNK cells by intracellular cytometry, but cells were first stimulated in culture for 3 weeks with IL-2 (Hanna et al., 2006 and personal communication). Other studies have used only ELISAs and may have been detecting IL-8 produced by macrophages contaminating dNK cell preparations (Vacca et al., 2008). We did observe dNK cells to degranulate in response to 721.221 target cells. A recent report found specifically dimeric HLA-G to modulate decidual leucocyte cytokines produced in response to 721.221 target cells (Li et al., 2009). We saw no effect on dNK degranulation of either HLA-G conformation, whereas PBNK cytotoxicity was inhibited, as is well established (Colonna et al., 1997; Perez-Villar et al., 1997; Navarro et al., 1999; Morel and Bellon, 2008). dNK cell responses to stimulation by NKp30 and NKp46 have been reported but were not investigated here (El Costa et al., 2008).

Our functional experiments can be summarized simply, in that no effect of HLA-G was detected on freshly isolated first trimester dNK cells. Thus, neither of the putative HLA-G receptors, LILRB1 or KIR2DL4, could be shown to play a role in regulating dNK function using common NK cell functional read-outs. Degranulation, IFN-γ and IL-8 production were measured on the basis of previous findings from our laboratory and others using PBNK or NK cell lines, clones or cytokine-stimulated cells derived from dNK. It is of course possible that freshly isolated ex vivo dNK cells respond differently from NK cells encountering EVT at the implantation site in vivo. This proviso would also apply, however, to experiments using PBNK or dNK after culture. A further complication is that HLA-G on EVT may well affect dNK differently compared with HLA-G on transfected 721.221 cells. Our study does highlight the difficulties in investigating function of tissue NK cells that are crucially different in phenotype and function from PBNK. A continued search for functional assays that reflect the in vivo function of dNK cells in the placental bed is therefore essential. It will also be interesting to learn the effects of HLA-G on decidual myelomonocytic cells present at the implantation site, which all express LILRB1. There is increasing evidence that LILRB1 stimulation is important in the development of tolerogenic dendritic cell responses. That HLA-G modulates the context in which trophoblast antigens are presented to establish a favourable maternal immunological response remains an attractive idea (Ristich et al., 2005; Tenca et al., 2005; Apps et al., 2007).

Authors' roles

R.A. performed the study design, experimental work and manuscript preparation. V.M., L.G., A.S., P.K., L.M., D.J., L.F. and R.T. contributed substantially to the design and/or completion of the cytometry, histology, genotyping and functional experiments. A.M. conceived this study as well as supervised its design, execution and presentation.

Funding

A.M. is supported by the Wellcome Trust, British Heart Foundation, Wellbeing of Women and the Centre for Trophoblast Research. R.A. is supported by a Next Generation fellowship from the Centre for Trophoblast Research, Cambridge.

Acknowledgements

We would like to thank the patients and clinicians, without whom this study would not have been possible, Dr Barbara Uchanska-Ziegler for providing Tu149 mAb and Dr Smita Kulkarni for the assay to genotype polymorphisms in the miR148-binding site.

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