Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes

Significance

Fetal extravillous trophoblasts (EVT) invade uterine tissue and interact with maternal immune cells during pregnancy. EVT express human leukocyte antigen-C (HLA-C) and -G (HLA-G). Although polymorphic HLA-C can elicit a maternal immune response, HLA-G has been associated with induction of immune tolerance. We have succeeded in isolating all maternal immune cell types as well as EVT from human placental tissue. These methods were used to elucidate the unique charateristics of EVT as well as their interaction with maternal immune cells. We demonstrate that EVT are specialized cells whose properties are not imitated by HLA‐G–expressing surrogate cell lines. Studies using primary EVT are crucial for understanding maternal–fetal tolerance and development of pregnancy complications such as preeclampsia and miscarriages.

Abstract

Invading human leukocyte antigen-G+ (HLA‐G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA‐G+ EVT and HLA‐G− villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA‐G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA‐G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25^HIFOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal–fetal tolerance, the properties of which are not imitated by HLA‐G–expressing surrogate cell lines.

During pregnancy invading fetal human leukocyte antigen-G+ (HLA-G+) extravillous trophoblasts (EVT) play a key role in the process of placental anchoring, opening of the uterine spiral arteries, and prevention of a maternal immune attack on foreign placental and fetal tissues. Failures of these processes have been postulated to play a role in miscarriages, preterm birth, and preeclampsia although little is understood about the mechanisms involved (1). Upon blastocyst implantation, the extraembryonic cells differentiate into several types of trophoblasts that have distinct functions and unique ways of interacting with maternal immune cells (1). The main types of trophoblasts include the MHC negative villous trophoblasts (VT) and the invading HLA-G+ EVT. VT, including cytotrophoblasts and syncytiotrophoblasts, form the placental floating villi and are the barrier between fetal cord blood and the maternal blood in the intervillous space.

HLA-G+ EVT, found at the tips of anchoring villi (columnar EVT), detach from these villous columns and adhere to extracellular matrix proteins, migrate through decidual tissue (interstitial EVT), and invade the uterine spiral arteries where they replace the endothelial cell layer (endovascular EVT). The factors required to promote differentiation and proliferation of EVT are unknown but may include interaction with extracellular matrix proteins and growth factors such as epidermal growth factor (EGF), leukemia inhibitory factor (LIF), bone morphogenetic factor-4 (BMP4), and fibroblast growth factor-4 (FGF4) (2–5). EVT invade decidual tissue and interact with maternal decidual natural killer cells (dNK), decidual macrophages (dMϕ), and decidual T cells (dT) (1, 6). EVT are difficult to study due to the low EVT numbers that can be obtained from tissue and the lack of proliferative capacity that precludes their expansion in vitro (7–9).

EVT express HLA-C, HLA-E, and HLA-G (but not HLA-A and HLA-B) and avoid immune rejection by maternal leukocytes (1). HLA-E and HLA-G have been shown to limit NK cytotoxicity (10, 11). HLA-G–expressing cells also have been shown to increase the secretion of cytokines such as interleukin-6 (IL-6) and IL-8 (6, 12, 13), to induce regulatory T cells (Treg) (14), and to modulate antigen-presenting cells by binding to the HLA-G receptor Leukocyte Ig-like receptor subfamily B member 1 (LILBR1, also known as ILT2) (13, 15). At term, pregnancy allogeneic paternal HLA-C expressed on EVT was correlated with maternal dT activation and induction of fetus-specific Treg (16). Furthermore, genetic association studies have shown that mothers who have the activating Killer Ig-like Receptor-2DS1 (KIR2DS1) are protected from pregnancy complication when its ligand, the C2 allo-type of HLA-C, is expressed by the fetus (17). From these data it may be postulated that immune activation by EVT is required to activate dNK to facilitate trophoblast invasion while preventing immune rejection by active induction of Treg.

Thus, far the potential of dNK, dT, and dMϕ to secrete cytokines or kill target cells has relied on the use of trophoblast-like cell lines (e.g., JEG3), target cell lines (e.g., 721.221 or K562), the transfectant 721.221/HLA-G, or stimulation with mitogens (e.g., phorbol myristate acetate, or PMA) or antibodies that target specific receptors (e.g., KIR2DS1 or CD3). These protocols underlined the numerous differences that exist between peripheral blood and decidual leukocytes (6, 13, 18, 19). dNK were shown to have decreased cytotoxicity and IFNγ secretion and increased production of cytokines such as IL-8, vascular endothelial growth factor (VEGF), granulocyte monocyte-colony stimulating factor (GM-CSF), and galectin-1 (1LGALS), compared with peripheral blood NK cells (pNK) (6, 13, 20–23). dMϕ, of which there are at least two subtypes, produce high levels of pro- as well as anti-inflammatory cytokines (19). dT are a heterogeneous population with increased levels of CD4+ Treg and CD8+ Effector-Memory T cells compared with peripheral blood (18, 24). However, thus far no study has systematically examined the immune response of dNK, dT, and dMϕ directly to purified VT or HLA-G+ EVT.

In the present study, a procedure was developed to obtain robust VT and EVT preparations as well as all major maternal decidual leukocyte types (dNK, dMϕ, CD4+, and CD8+ dT) from the same pregnancy. These preparations have been used to study the properties of EVT by flow cytometry, microarray, and gene set enrichment analysis. In addition, cocultures of EVT and decidual leukocytes were established to investigate their contributions to the establishment and/or maintenance of maternal–fetal tolerance in human pregnancy.

Results

Isolation of VT and EVT.

Cell preparations of human first-trimester villous tissue (Materials and Methods), were directly stained for EGF Receptor 1 (EGFR1, also known as ERBB1), HLA-G, and the common leukocyte antigen (CD45) and analyzed by flow cytometry (Fig. 1A). FACS analysis demonstrated a median of ∼9% CD45-HLA-G+EGFR1^dim EVT (with some samples as high as 20%), ∼77% CD45-HLA-G-EGFR1+ VT, and ∼7% CD45+ leukocytes (Fig. 1B). The fraction of EVT did not vary with gestational age (SI Appendix, Fig. S1). However, the percentage of CD45-HLA-G-EGFR1+ VT decreased whereas CD45+ cells increased with gestational age (SI Appendix, Fig. S1). The yield was 0.3–7.4 × 10⁵ EVT and 0.4–3.3 × 10⁶ VT per preparation (SI Appendix, Table S1); the range is due to gestational age and size of the tissue. Both EVT and VT expressed the trophoblast marker cytokeratin-7 but not the stromal cell marker vimentin (SI Appendix, Fig. S2A). FACS analysis confirmed HLA-C expression on EVT, whereas HLA-E was relatively low. VT were MHC-negative (SI Appendix, Fig. S2B).

Fig. 1.

Trophoblast preparations. (A) Representative FACS plots of trophoblast preparations. A live gate (R1) was set in the Forward-Side Scatter plot and CD45+ leukocytes were excluded with gate R2. Subsequently, EGFR1–FITC and HLA-G–PE were plotted, and separation of EVT (CD45-EGFR1^dimHLA-G+) and VT (CD45-HLA-G-EGFR1+) is depicted. (B) Percentage of CD45+ leukocytes (within live gate), CD45-EGFR1^dimHLA-G+ EVT, and CD45-HLA-G-EGFR1+ VT (both within live gate and CD45− fraction) of the trophoblast preparations. (C) HLA-G expression on freshly isolated trophoblast preparations and preparations cultured for 2 d on fibronectin. (D) Representative FACS plots of HLA-G and EGFR1 expression on FACS-sorted CD45-HLA-G+ EVT, CD45-HLA-G-EGFR1+ VT, and the remaining CD45-HLA-G-EGFR1− cells that were cultured on fibronectin for 2 d. (E) EGFR1, EGFR2, and LIFR expression on freshly isolated EVT and EVT cultured for 2 d on fibronectin.

Up-Regulation of HLA-G and Growth Factor Receptors on EVT by Culture on Fibronectin.

When trophoblast isolates were cultured on fibronectin for 2 days and afterward washed and harvested, a marked increase in the percentage of HLA-G+ cells as well as the intensity of HLA-G on the cells was observed (Fig. 1C). After FACS sorting for CD45-HLA-G+EGFR1^dim EVT (Fig. 1A), 98% of cells in the EVT fraction were HLA-G+, whereas the CD45-HLA-G-EGFR1+ VT and the remaining CD45-HLA-G-EGFR1− cells were also at 99% purity (Fig. 1D). For the CD45-HLA-G-EGFR1+ VT fraction, very few adherent cells were found, and neither the adherent nor the nonadherent fractions showed HLA-G expression after culture on fibronectin. Further studies are needed to establish whether ligands present in fibrinoid material in decidua and Matrigel that can engage additional integrins have distinct effects on VT and EVT phenotype and capacity to proliferate or differentiate as found previously (25). EVT have been shown to express many receptors for growth factors that can play a role in trophoblast proliferation and/or differentiation. The expression of EGFR2 (also known as CD340 or ERBB2), LIFR, and, at low level, EGFR1 on EVT is confirmed, and, moreover, a marked increase in expression of EGFR2 and LIFR, but not EGFR1 was observed after culture on fibronectin (Fig. 1E) (2). VT expressed higher levels of EGFR1 and did not express EGFR2 or LIFR. Culture on fibronectin did not change the expression on VT (SI Appendix, Fig. S3A). In addition, trophoblast isolates were cultured on fibronectin for 1 or 2 days and stained for the proliferation marker Ki67, but no proliferation indicated by Ki67+ cells was observed (SI Appendix, Fig. S3B). Total trophoblast preparations were also labeled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and analyzed at day 3, but no proliferation was observed (SI Appendix, Fig. S3C).

VT and EVT Have Very Distinct Transcriptional Profiles.

RNA was obtained from seven patient-paired and freshly isolated VT and EVT from the HLA-G–expressing cell line JEG3 on two different occasions and from four different decidual stromal cell (DSC) cultures. The 20 RNA samples were hybridized independently to Human Genome Affymetrix U133 plus 2.0 chips. Unsupervised hierarchical cluster analysis resulted in a dendrogram where each cell type formed a distinct cluster (Fig. 2A). As expected, DSC formed a separate branch from the other three cell types, whereas EVT were closer to JEG3 than to VT.

Fig. 2.

Expression profiling of EVT and VT. (A) Unsupervised hierarchical cluster analysis results in a dendrogram where each distinct cell type (VT, EVT, JEG-3, and DSC) forms a separate cluster. Volcano plots were generated based on the mean expression value of an individual probe’s fold change and the P value associated with reproducibility of these changes between (B) VT and EVT and (C) EVT and JEG-3. The unique gene signatures based on a more than fourfold difference for VT (340 probes, blue dots) and EVT (328 probes, red dots) are highlighted.

To evaluate the genomic differences that distinguish VT and EVT, a volcano plot was generated based on the mean expression value of an individual probe’s fold change and the P value associated with reproducibility of these changes between the VT and EVT (Fig. 2B). This identified 2,252 probes up-regulated in VT and 1,953 probes up-regulated in EVT with a twofold difference and indicated that VT and EVT are very distinct cell types. To generate more stringent gene signatures for VT and EVT, genes were selected based on fourfold differential expression present in all seven patient pairs. This identified 340 probes (encoding 221 unique genes after exclusion of duplicate genes and uncharacterized loci) that were up-regulated in VT (Fig. 2B, blue dots) whereas 328 probes (encoding 210 unique genes) were up-regulated in EVT (Fig. 2B, red dots). The annotated probe lists are included in Datasets S1 and S2. The gene signature of EVT was overlaid on volcano plots that compare JEG3 vs. EVT and DSC vs. EVT and showed that the majority of the genes identified for EVT were absent in JEG3 and DSC (Fig. 2C and SI Appendix, Fig. S4A). The gene signatures for HLA-G+ EVT generated by two previous studies were overlaid on the volcano plots and demonstrated the similarity of the findings in both studies although distinct probes were also identified (SI Appendix, Fig. S4 B and C) (26, 27). Interestingly, the gene list for EGFR1+ VT (26) demonstrated a clear match whereas the signature for tumor-associated calcium signal transducer-2 (TACSTD2)–expressing VT (27) did not match. The trophoblast isolation procedure used to isolate TACSTD2+ VT was different in trypsin digestion time (8 vs. 30 min), density gradient (ficoll vs. percoll), and selection marker (EGFR1+ vs. TACSTD2+). This may indicate that EGFR1+ VT and TACSTD2+ VT are distinct subtypes of VT.

Detailed analysis of transcriptional differences between EVT and VT with respect to growth factors and cytokines, receptors for growth factors and cytokines, cell adhesion molecules, and ECM components are presented as heat maps in SI Appendix, Fig. S5. Here, focus is placed on the integrin family of cell adhesion molecules because of their possible role in up-regulation of HLA-G on EVT after culture on fibronectin (Fig. 1C) and because of an extensive literature on integrins on the development of EVT (28). In addition to the integrin subunits that are differentially expressed (ITGA5 on EVT and ITGB5 and ITGB8 on VT), the α- and β-subunits of a number of integrin heterodimers were expressed on both trophoblast cells (SI Appendix, Fig. S6). Integrin α₅β₁, a fibronectin receptor, was uniquely expressed by EVT and may contribute to the up-regulation of HLA-G. Integrin α_Vβ₅ and α_Vβ₈, both vitronectin receptors, are uniquely expressed on VT. Three integrins are expressed by both EVT and VT: α₆β₄ and α₆β₁ (both laminin receptors) and α_Vβ₁ (a vitronectin receptor). FACS and microarray data demonstrated ITGA6 and ITGB4 expression on VT and EVT. However, the expression level on VT was increased and may explain the difference found in a previous study (29). The ligands for integrins are present in Matrigel as well as in the fibrinoid material in the maternal decidua (25).

Gene Set Enrichment Analysis.

To identify the functional differences that reflect the transcriptional differences between EVT and VT, a Gene Set Enrichment Analysis (GSEA) was performed. GSEA is a computational method that uses an a priori-defined set of genes and determines which “functional” sets of genes are up-regulated. GSEA software was used to generate a list of functional gene sets that are specifically overrepresented in VT or EVT. Interestingly, in EVT, 14 functional gene sets of the 20 most significantly enriched gene sets were associated with direct immune and lymphocyte activation (SI Appendix, Table S2). The core genes of these immune-activation–related pathways are cytokines such as Epstein–Barr virus-induced gene-3 (EBI3), tumor growth factor beta-1 (TGF-β1), TGF-β2, IL-8, and the cell-surface molecule CD276 (B7-H3) and cytotoxic and regulatory T-cell molecule (CRTAM), which can all directly influence lymphocyte binding and are potent inhibitors of lymphocyte activation (SI Appendix, Fig. S7). EBI3 is known to dimerize with two α-chains, IL-12(p28) and IL-12(p35), to form the immune-suppressive cytokines IL-27 and IL-35 (30). However, neither of these IL-12 α-chains are expressed by EVT. How EIB3 functions in EVT and whether EBI3 can form homo-dimers or pairs with an unknown α-chain remain to be determined. The functional gene sets enriched in VT were less significant (increased false discovery rate) and more diverse in function (SI Appendix, Table S3).

Coculture of EVT with Sample Matched Maternal Leukocytes.

To evaluate the implications of the striking immune-activating and -regulating potential of EVT demonstrated in the GSEA, VT- and EVT-enriched cocultures with all major maternal leukocyte subsets (dNK, dMɸ, CD4+, and CD8+ dT cells) were established. For this sample, matched trophoblast and decidual leukocytes or trophoblasts and peripheral blood leukocytes from unrelated nonpregnant donors were used. The FACS sorting strategy for all cell types can be found in SI Appendix, Fig. S8. Microscopy images revealed that EVT adhere to fibronectin with a distinct, large, spread-out morphology and interact with multiple other EVT. Furthermore, HLA-G+ EVT with various sizes and morphology are found in the cell cultures whereas VT are homogenous, small, rounded cells that do not adhere to fibronectin (SI Appendix, Fig. S9A). Light microscopy also revealed clustering of NK, CD4+, and CD8+ T cells around the large EVT. Multiple NK or T cells can interact with one EVT, either at the cell body or at filopodia-like structures that spread out from the EVT cell body (SI Appendix, Fig. S9B). No signs of cytolysis of EVT were observed in any of the trophoblast–leukocyte cocultures.

EVT Increase the Fraction of CD4+CD25^HIFOXP3+CD45RA+ Treg.

Treg are major regulators of immune responses at the maternal–fetal interface (18, 31). To assess the influence of EVT on Treg, CD4+ dT and peripheral blood CD4+ T cells (CD4+ pT) were cultured alone and together with VT and EVT for 3 days. T cells were harvested and analyzed for the presence of CD4+CD25^HIFOXP3+ Treg. Coculture of CD4+ dT and CD4+ pT with EVT significantly increased the percentage of CD4+CD25^HIFOXP3+ Treg. In addition, EVT significantly increased the expression level (mean fluorescence intensity, or MFI) of FOXP3 within the CD4+CD25^HIFOXP3+ Treg after coculture with CD4+ pT (Fig. 3 A–C). The FOXP3 level in CD4+CD25^HI dT was already very high and did not further increase by coculture with EVT. When CD4+CD25^HI-depleted CD4+ pT (containing both naive CD4+CD25− and activated CD4+CD25^DIM T cells but not CD4+CD25^HI Treg) were added to EVT, no induction of CD4+CD25^HIFOXP3+ Treg was observed (SI Appendix, Fig. S10B). In contrast, when both total CD4+ pT and purified CD4+CD25^HI pT were cocultured with EVT, a marked increase in CD4+CD25^HIFOXP3+ Treg compared with CD4+ or CD4+CD25^HI pT cultured alone was seen (SI Appendix, Fig. S10 A and C). Thus, EVT specifically increased FOXP3 expression in CD4+CD25^HI cells as opposed to a de novo induction of FOXP3 in CD4+CD25− T cells. CD4+ pT or CD4+CD25^HI pT were labeled with CFSE and incubated with or without EVT, VT, or anti-CD3/CD28 beads. Only the pT cells incubated with anti-CD3/CD28 beads showed proliferation at day 3, demonstrating that the increase in FOXP3 expression is not due to increased proliferation of these cells (SI Appendix, Fig. S10D). Furthermore, when purified pCD4+CD25^HI cells were cocultured with EVT, a significant increase of CD4+CD25^HIFOXP3+ CD45RA+ Treg, previously described to be resting Treg (32), was observed in the EVT but not in the VT cultures (Fig. 3 D and E). Thus, EVT but not VT directly influence the generation of CD4+CD25^HIFOXP3+CD45RA+ resting Treg.

Fig. 3.

Coculture with EVT increases the percentage of CD4+FOXP3+ Treg. (A) Representative FACS plots of CD25 and FOXP3 expression of CD4+ pT in the absence of stimulation or after coculture with VT- or EVT-enriched preparations. (B) The percentage of FOXP3+ cells within total CD4+ pT and dT and (C) MFI of FOXP3 expression within CD4+CD25^HIFOXP3+ pT and dT cocultured with or without VT- or EVT-enriched preparations. (D) Representative FACS plots of CD45RA and FOXP3 within CD4+CD25hi pT cocultured with or without VT- or EVT-enriched preparations. (E) The percentage of FOXP3+CD45RA− and (F) FOXP3+CD45RA+ cells within each group.

EVT Do Not Elicit Specific Cytokine Responses by Maternal dNK, dMϕ, or dT Cells.

Cell culture supernatants were harvested from EVT-enriched and leukocyte cocultures at day 1 (NK) and day 3 (Mɸ and T cells), and cytokines were analyzed by using a multiplex cytokine assay. As a negative control, all cell types were cultured alone and as positive controls with 721.221 (221) MHC class I negative target cells, with 721.221/HLA-G (221.HLA-G) or with PMA to activate NK or anti-CD3/CD28 to activate T cells.

Natural killer cells.

Upon stimulation with PMA (but not with 221 cells), pNK secreted significantly higher levels of the proinflammatory cytokines granulocyte monocyte–colony-stimulating factor (GM-CSF), IFN-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα) compared with dNK (Fig. 4 A–C). Reciprocally, upon stimulation with 221 cells (but not with PMA), dNK secreted significantly higher levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) than pNK (Fig. 4 D and E), confirming previous studies (6, 13, 22). Interestingly, expression of HLA-G on 221 cells reduced IL-6 secretion in dNK. Thus, HLA-G expression may play a direct role in suppression of at least one proinflammatory factor. However, HLA-G expression on 221 cells did not alter secretion of VEGF, an important factor that contributes to vascular remodeling. Most surprisingly, when dNK were cocultured with VT- or EVT-enriched preparations, little or no effect on cytokine secretion was observed. IL-6 was slightly elevated but no secretion of GM-CSF, IFNγ, TNFα, VEGF, or IL-17 was induced in dNK (or in pNK) (Fig. 4 A–F). Thus, PMA, 221 cells, and 221/HLA-G cells used as stimulators revealed differences in pNK and dNK cytokine responses, but this did not reflect VT or EVT responses. Thus, to understand the unique properties of dNK and EVT–dNK interactions, primary EVT need to be included in further studies. Secretion of IL-8 (neutrophil chemotactic factor) and galectin-1 (involved in suppression of activated T cells) was significantly higher in dNK compared with pNK (Fig. 4 G and H). However, the secretion of IL-8 and galectin-1 by dNK was not amplified by PMA, VT, or EVT stimulation. Thus, dNK (but not pNK) constitutively secreted high levels of IL-8 and galectin-1. Prior activation or differentiation of dNK in vivo may be responsible for inducing IL-8 and galectin-1. Supernatants were also analyzed for the anti-inflammatory cytokines IL-10 and IL-22, but their secretion was not observed. Furthermore, all dNK and pNK were analyzed for the expression of KIR2DS1. No differences were found in cytokine responses between dNK or pNK from KIR2DS1+ and KIR2DS1− individuals toward any of the target cells, including EVT.

Fig. 4.

Cytokine secretion profiles of pNK and dNK during coculture with VT and EVT. pNK (blue) and dNK (red) were incubated alone or with PMA, 221, 221/HLA-G, and VT- or EVT-enriched preparations for 18 h. Cell culture supernatants were analyzed for (A) GM-CSF, (B) IFNγ, (C) TNFα, (D) VEGF, (E) IL-6, (F) IL-17A, (G) IL-8, and (H) galectin-1. VT- or EVT-enriched preparations cultured alone secreted IL-6 and IL-8 (E and G, depicted by gray dots), but did not secrete any of the other cytokines.

Macrophages.

As demonstrated previously, dMϕ constitutively secrete both the pro- and anti-inflammatory cytokines IL-10, IL-1β, TNFα, and IL-6 (19). However, in contrast to stimulation with lipopolysaccharide (LPS) or LPS/IFN-γ (19), stimulation of dMϕ or monocytes VT- or EVT-enriched preparations did not change the secretion of any of these cytokines (SI Appendix, Fig. S11). IL-12(p70), a potent T-cell–stimulating cytokine, and IL-33, a member of the IL-1 family recently implicated in enhancing proliferation of primary trophoblasts (33) and promotion of Treg survival (34), were not detected in any of the dMϕ or monocyte cultures.

T cells.

CD4+ and CD8+ dT and pT were incubated with or without anti-CD3/CD28 beads and VT- or EVT-enriched preparations for 3 days. Although both CD4+ and CD8+ dT and pT had a profound IFNγ response to anti-CD3/CD28 beads, none of the T cells secreted IFNγ in response to VT or EVT (SI Appendix, Fig. S12).

Discussion

EVT are found in at least four different locations in the pregnant uterus, i.e., as the cells at the tips of anchoring villi in cell columns (columnar EVT), as interstitial EVT within the decidua in contact with maternal leukocytes, as endovascular EVT within the uterine spiral arteries in contact with the vascular endothelium, and as cells in chorionic tissue directly adjacent to the maternal decidual or parietal surface (chorionic EVT). In interpreting the present data, it is important to note the origin of the EVT studied here. These cells were obtained from first-trimester villous tissue separated from the decidua. They largely represent cells obtained from cell columns at the tips of anchoring villi. Interstitial EVT and endovascular EVT would represent a very small population—too small to be isolated under the present circumstances. Some data suggest that these EVT are all distinct in expression of different molecules related to distinct functions (28, 35). In that context, the present preparation may represent EVT at an intermediate level of differentiation. Culture on fibronectin-enhanced HLA-G expression as well as expression of EGFR2 and LIFR, presumably representing a further differentiation state. The up-regulation of HLA-G on EVT by culture on fibronectin during the preparations is likely to involve the interaction of fibronectin with an integrin on EVT, probably α₅β₁ that is prominently expressed by these cells (25, 28).

Importantly, coculture of CD4+ T cells with EVT increased the proportion of FOXP3 as well as the expression level of FOXP3 within Treg, suggesting that EVT may directly enhance Treg function. Initial experiments demonstrate that the increase of FOXP3+ is not dependent on proliferation but involves conversion of CD4+CD25^HI FOXP3− cells into FOXP3+ Treg. In addition, EVT, but not VT, significantly increased the proportion of CD4+CD25^HIFOXP3+CD45RA+ resting Treg. The increase in resting Treg after coculture with EVT demonstrates that EVT directly contributes to augmentation of the maternal Treg pool at the maternal–fetal interface. Although the mechanisms responsible remain unknown, the increase in FOXP3 and proportion of Treg may contribute to suppression of maternal immune responses directed to polymorphic fetal HLA-C molecules expressed on EVT (16). However, the antigen specificity of the Treg at the fetal–maternal interface as well as whether cell contact and/or soluble factors are required to promote Treg are questions of key importance to understanding maternal–fetal tolerance.

Gene chip analysis on RNA obtained from seven patient-paired VT and EVT preparations was performed. In contrast to two recent studies (26, 36), the VT and EVT preparations were highly purified and used directly after isolation without in vitro culture to minimize transcriptional changes that may occur with cell culture. Interestingly, the signature for TACSTD2+ VT (27) did not match the transcriptional signature for EGFR1+ VT and may indicate that these are distinct subtypes of VT. Similarly, confocal imaging suggests that multiple HLA-G+ EVT subtypes with various sizes and morphology are found in the preparations. Previous studies using different isolation protocols may have selected trophoblasts with distinct characteristics (37). Further effort needs to be made to characterize distinct VT and EVT types present in placental tissue. The gene set enrichment analysis revealed a striking immune-activating potential for EVT and identified genes able to directly modulate trophoblast–lymphocyte interactions. The core genes identified by the GSEA include known cytokines expressed by EVT such as EBI3, TGF-β1, TGF-β2, Inhibin beta A (INHB), and IL-8 and molecules such as CD276 (B7-H3) and CRTAM that can directly influence NK and T-cell binding to EVT. Furthermore, the EVT gene signature revealed that EVT express a variety of cytokine receptors that may render them responsive to cytokines and chemokines secreted by leukocytes such as IL-10, IL-2, IL-1, MIP-1α, RANTES, MCP-3, GM-CSF, M-CSF, and G-CSF.

Previous studies have suggested that EVT are derived from VT at the end tips of the villous tree. In this study, 4,205 probes that are differentially expressed between EVT and VT were identified (2,252 probes are overrepresented in EVT and 1,953 in VT with a twofold difference). Furthermore, the GSEA and the unique VT and EVT gene signatures revealed major differences in cellular functions and expression of transcription factors. These large differences may indicate that VT and EVT are derived from a distinct cellular origin. Alternatively, EVT precursor cells or trophoblast stem cells at the end tips of the villous tissue may give rise to fully differentiated EVT. The proliferation and/or outgrowth of EVT observed by BrDU incorporation in placental explant cultures (7) may represent a growth burst derived from specialized precursor cells that differentiate into EVT or proliferation by HLA-G+ cells at the villous tip. Alternatively, VT may include distinct cell types (e.g., EGFR1+ and TACSTDC2+) of which some can and others cannot differentiate into HLA-G–expressing cells. Furthermore, cell culture conditions used here may lack additional growth factors, integrin ligands, or oxygen/CO₂ levels required for trophoblast differentiation.

Because of the difficulties in obtaining human HLA-G+ EVT, many studies have attempted to generate human HLA-G+ EVT lines. However, careful assessment of the cell lines and their MHC expression demonstrates that these cell lines do not express a similar MHC profile compared with EVT (38). The closest model cell line for EVT and its atypical HLA-C, HLA-E, and HLA-G expression remains the choriocarcinoma cell line JEG-3. The transcriptional profiles of JEG-3 differed from EVT by 1,475 probe sets, many representing key functions of EVT, e.g., cytokine production, growth factor receptors, ECM components and enzymes, and MHC regulatory molecules. In this study, robust and highly purified VT and EVT preparations were obtained and used to establish cocultures with decidual leukocytes. Most interestingly, in contrast to previous studies that used surrogate HLA-G+ cell lines, HLA-G+ EVT did not elicit a profound cytokine response by dNK, pNK, dMϕ, monocytes, and CD4+ or CD8+ dT or pT, even though the distinct cytokine secretion profiles of dNK and pNK in response to stimulation either by 221 cells or 221/HLA-G target cells or by PMA was confirmed.

Thus, careful and systematic validation of EVT–leukocyte interactions needs to be carried out by using primary HLA-G+ EVT to understand the unique contribution of EVT to the decidual immune response in human pregnancy. The surrogate cell lines presently available are not adequate for this purpose. Studies on the role of primary EVT in maternal–fetal tolerance will be crucial to understanding the development of pregnancy complications such as preterm delivery and preeclampsia.

Materials and Methods

Discarded human placental and decidual material was obtained from women undergoing elective pregnancy termination at 6–12 wk at a local reproductive health clinic. Peripheral blood leukocytes were isolated from discarded leukopacks from healthy volunteer blood donors from the Massachusetts General Hospital (Boston, MA). All of the human tissue used for this research was de-identified, discarded clinical material. The Committee on the Use of Human Subjects (the Harvard Institutional Review Board) determined that this use of placental and decidual material is not human subjects research. Decidual and villous tissues were macroscopically identified and separated. Trophoblasts were isolated as described previously (26, 39). In short, villous tissue was scraped from the basal membrane and digested for 8 min at 37 °C with trypsin (2 g/L) and EDTA (0.2 g/L). Trypsin was quenched with F12 medium with 1.0 mL/1 mL newborn calf serum and penicillin/streptomycin (Gibco) and filtered over a gauze mesh. Filtrate was layered on Ficoll (GE Healthcare) for density gradient centrifugation (20 min at 800 × g) and thereafter incubated for 20 min at 37 °C in a culture dish for removal of macrophages. Nonadherent cells were collected and directly stained for flow cytometric analysis or FACS sorting.

Decidual tissue was washed, minced, and digested with 0.1 mg/mL collagenase IV and 0.01 mg/mL DNase I (Sigma) shaking in a water bath for 1 h at 37 °C. Released lymphocytes were filtered through 100-, 70-, and 40-μm sieves (BD Labware). Lymphocytes were dissolved in 20 mL 1.023g/mL Percoll (GE Healthcare) and layered on 10 mL 1.080g/mL and 12.5 mL 1.053g/mL Percoll for density gradient centrifugation (30 min at 800 × g). Lymphocytes were isolated from the 1.080 to 1.053 g/mL interface and dMϕ from the 1.053 to 1.023 g/mL interface. Cells were washed and directly stained for flow cytometric analysis or FACS sort (SI Appendix, Fig. S8). pNK, pT, and monocytes from leucopacks were isolated with RosetteSep (StemCell Technologies). For all leukocyte types, >95% purity was obtained.

Cocultures, RNA isolation, microarray hybridization and analysis, flow cytometry, cytokine analysis, and imaging and statistics used are described in SI Appendix.