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

Tamara Tilburgsa,1, Ângela C. Crespoa,b, Anita van der Zwana, Basya Rybalova, Towfique Rajc, Barbara Strangerd, Lucy Gardnere, Ashley Moffette, and Jack L. Stromingera,1

aDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; bPhD Program in Experimental Biology and Biomedicine, Center for Neurosciences and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal; cDepartment of Neurology, Brigham and Women’s Hospital, Boston, MA 02115; dInstitute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637; and eDepartment of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom

Contributed by Jack L. Strominger, April 24, 2015 (sent for review February 27, 2015)

Invading human leukocyte antigen-G+ (HLA‐G+) extravillous tro- EVT numbers that can be obtained from tissue and the lack of phoblasts (EVT) are rare cells that are believed to play a key role in proliferative capacity that precludes their expansion in vitro (7–9). the prevention of a maternal immune attack on foreign fetal tis- EVT express HLA-C, HLA-E, and HLA-G (but not HLA-A sues. Here highly purified HLA‐G+ EVT and HLA‐G− villous tropho- and HLA-B) and avoid immune rejection by maternal leukocytes blasts (VT) were isolated. Culture on fibronectin that EVT encounter (1). HLA-E and HLA-G have been shown to limit NK cytotox- on invading the uterus increased HLA‐G, EGF-Receptor-2, and LIF- icity (10, 11). HLA-G–expressing cells also have been shown to Receptor expression on EVT, presumably representing a further dif- increase the secretion of cytokines such as interleukin-6 (IL-6) ferentiation state. Microarray and functional gene set enrich- and IL-8 (6, 12, 13), to induce regulatory T cells (Treg) (14), and ment analysis revealed a striking immune-activating potential for to modulate antigen-presenting cells by binding to the HLA-G EVT that was absent in VT. Cocultures of HLA‐G+ EVT with sample receptor Leukocyte Ig-like receptor subfamily B member 1 matched decidual natural killer cells (dNK), macrophages, and CD4+ (LILBR1, also known as ILT2) (13, 15). At term, pregnancy al- and CD8+ T cells were established. Interaction of EVT with CD4+ logeneic paternal HLA-C expressed on EVT was correlated with T cells resulted in increased numbers of CD4+CD25HIFOXP3+CD45RA+ maternal dT activation and induction of fetus-specific Treg (16). Furthermore, genetic association studies have shown that mothers resting regulatory T cells (Treg) and increased the expression level of who have the activating Killer Ig-like Receptor-2DS1 (KIR2DS1) the Treg-specific transcription factor FOXP3 in these cells. However, are protected from pregnancy complication when its ligand, the EVT did not enhance cytokine secretion in dNK, whereas stimulation C2 allo-type of HLA-C, is expressed by the fetus (17). From these of dNK with mitogens or classical natural killer targets confirmed the data it may be postulated that immune activation by EVT is re- distinct cytokine secretion profiles of dNK and peripheral blood NK quired to activate dNK to facilitate invasion while cells (pNK). EVT are specialized cells involved in maternal–fetal tol- ‐ – preventing immune rejection by active induction of Treg. erance, the properties of which are not imitated by HLA G express- Thus, far the potential of dNK, dT, and dMϕ to secrete cy- ing surrogate cell lines. tokines 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), Treg | FOXP3 | NK cell | human | pregnancy the transfectant 721.221/HLA-G, or stimulation with mitogens

uring pregnancy invading fetal human leukocyte antigen-G+ Significance D(HLA-G+) extravillous trophoblasts (EVT) play a key role in the process of placental anchoring, opening of the uterine Fetal extravillous trophoblasts (EVT) invade uterine tissue and spiral arteries, and prevention of a maternal immune attack on interact with maternal immune cells during pregnancy. EVT foreign placental and fetal tissues. Failures of these processes have express human leukocyte antigen-C (HLA-C) and -G (HLA-G). been postulated to play a role in miscarriages, preterm birth, and Although polymorphic HLA-C can elicit a maternal immune preeclampsia although little is understood about the mechanisms response, HLA-G has been associated with induction of im- involved (1). Upon blastocyst implantation, the extraembryonic mune tolerance. We have succeeded in isolating all maternal cells differentiate into several types of trophoblasts that have dis- immune cell types as well as EVT from human placental tissue. tinct functions and unique ways of interacting with maternal im- These methods were used to elucidate the unique charateristics mune cells (1). The main types of trophoblasts include the MHC of EVT as well as their interaction with maternal immune cells. + negative villous trophoblasts (VT) and the invading HLA-G We demonstrate that EVT are specialized cells whose properties EVT. VT, including and , are not imitated by HLA‐G–expressing surrogate cell lines. form the placental floating villi and are the barrier between fetal Studies using primary EVT are crucial for understanding maternal– cord blood and the maternal blood in the intervillous space. + fetal tolerance and development of pregnancy complications HLA-G EVT, found at the tips of anchoring villi (columnar such as preeclampsia and miscarriages. EVT), detach from these villous columns and adhere to extra-

cellular matrix proteins, migrate through decidual tissue (inter- Author contributions: T.T. and J.L.S. designed research; T.T., Â.C.C., A.v.d.Z., and B.R. stitial EVT), and invade the uterine spiral arteries where they INFLAMMATION

performed research; T.R., B.S., L.G., and A.M. contributed new reagents/analytic tools; IMMUNOLOGY AND replace the endothelial cell layer (endovascular EVT). The fac- T.T. and Â.C.C. analyzed data; and T.T. and J.L.S. wrote the paper. tors required to promote differentiation and proliferation of EVT Conflict of interest statement: J.L.S. is a consultant for King Abdulaziz University, Jeddah, are unknown but may include interaction with extracellular ma- Saudi Arabia. trix proteins and growth factors such as epidermal growth factor Data deposition: Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/ (EGF), leukemia inhibitory factor (LIF), bone morphogenetic arrayexpress) under accession no. E-MTAB-3217. factor-4 (BMP4), and fibroblast growth factor-4 (FGF4) (2–5). 1To whom correspondence may be addressed. Email: [email protected] or EVT invade decidual tissue and interact with maternal decidual [email protected]. natural killer cells (dNK), decidual macrophages (dMϕ), and de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cidual T cells (dT) (1, 6). EVT are difficult to study due to the low 1073/pnas.1507977112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1507977112 PNAS | June 9, 2015 | vol. 112 | no. 23 | 7219–7224 Downloaded by guest on October 1, 2021 (e.g., phorbol myristate acetate, or PMA) or antibodies that tar- A R2 get specific receptors (e.g., KIR2DS1 or CD3). These protocols underlined the numerous differences that exist between peripheral CD45+ EVT 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 HLA-G factor (VEGF), granulocyte monocyte-colony stimulating factor SSC VT (GM-CSF), and galectin-1 (1LGALS), compared with peripheral blood NK cells (pNK) (6, 13, 20–23). dMϕ,ofwhichthereareat FSC CD45 EGFR1 least two subtypes, produce high levels of pro- as well as anti-in- B C flammatory cytokines (19). dT are a heterogeneous population − IgG + + − fresh with increased levels of CD4 Treg and CD8 Effector-Memory − day 2 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+,andCD8+ dT) from the same pregnancy. These preparations have been used to D CD45-HLA-G+EGFRdim CD45-HLA-G-EGFR1+ CD45-HLA-G-EGFR1- study the properties of EVT by flow cytometry, microarray, and gene set enrichment analysis. In addition, cocultures of EVT and 98% 1% 1% decidual leukocytes were established to investigate their contri- butions to the establishment and/or maintenance of maternal– fetal tolerance in human pregnancy. 2% 99% HLA-G 0% Results

Isolation of VT and EVT. Cell preparations of human first-trimester EGFR1 villous tissue (Materials and Methods), were directly stained for E − IgG EGF Receptor 1 (EGFR1, also known as ERBB1), HLA-G, and − fresh the common leukocyte antigen (CD45) and analyzed by flow − day 2 cytometry (Fig. 1A). FACS analysis demonstrated a median of ∼9% CD45-HLA-G+EGFR1dim 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 per- EGFR1 EGFR2 LIFR centage of CD45-HLA-G-EGFR1+ VT decreased whereas CD45+ cells increased with gestational age (SI Appendix,Fig.S1). Fig. 1. Trophoblast preparations. (A) Representative FACS plots of tropho- – × 5 – × 6 blast preparations. A live gate (R1) was set in the Forward-Side Scatter plot The yield was 0.3 7.4 10 EVT and 0.4 3.3 10 VT per + – SI Appendix and CD45 leukocytes were excluded with gate R2. Subsequently, EGFR1 preparation ( , Table S1); the range is due to gesta- FITC and HLA-G–PE were plotted, and separation of EVT (CD45-EGFR1dimHLA- tional age and size of the tissue. Both EVT and VT expressed the G+) and VT (CD45-HLA-G-EGFR1+)isdepicted.(B) Percentage of CD45+ trophoblast marker cytokeratin-7 but not the marker leukocytes (within live gate), CD45-EGFR1dimHLA-G+ EVT, and CD45-HLA-G- vimentin (SI Appendix,Fig.S2A). FACS analysis confirmed HLA-C EGFR1+ VT (both within live gate and CD45− fraction) of the trophoblast expression on EVT, whereas HLA-E was relatively low. VT were preparations. (C) HLA-G expression on freshly isolated trophoblast prepara- MHC-negative (SI Appendix,Fig.S2B). tions and preparations cultured for 2 d on fibronectin. (D) Representative FACS plots of HLA-G and EGFR1 expression on FACS-sorted CD45-HLA-G+ Up-Regulation of HLA-G and Growth Factor Receptors on EVT by EVT, CD45-HLA-G-EGFR1+ VT, and the remaining CD45-HLA-G-EGFR1− cells Culture on Fibronectin. When trophoblast isolates were cultured that were cultured on fibronectin for 2 d. (E) EGFR1, EGFR2, and LIFR ex- on fibronectin for 2 days and afterward washed and harvested, a pression on freshly isolated EVT and EVT cultured for 2 d on fibronectin. 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+EGFR1dim EVT (Fig. 1A), the proliferation marker Ki67, but no proliferation indicated by + SI Appendix B 98% of cells in the EVT fraction were HLA-G+, whereas the Ki67 cells was observed ( ,Fig.S3 ). Total trophoblast CD45-HLA-G-EGFR1+ VT and the remaining CD45-HLA-G- preparations were also labeled with the fluorescent dye carboxy- EGFR1− cells were also at 99% purity (Fig. 1D). For the CD45- fluorescein succinimidyl ester (CFSE) and analyzed at day 3, but no HLA-G-EGFR1+ VT fraction, very few adherent cells were proliferation was observed (SI Appendix,Fig.S3C). found, and neither the adherent nor the nonadherent fractions showed HLA-G expression after culture on fibronectin. Further VT and EVT Have Very Distinct Transcriptional Profiles. RNA was studies are needed to establish whether ligands present in fi- obtained from seven patient-paired and freshly isolated VT and brinoid material in and Matrigel that can engage ad- EVT from the HLA-G–expressing cell line JEG3 on two dif- ditional have distinct effects on VT and EVT phenotype ferent occasions and from four different decidual stromal cell and capacity to proliferate or differentiate as found previously (DSC) cultures. The 20 RNA samples were hybridized indepen- (25). EVT have been shown to express many receptors for growth dently to Human Genome Affymetrix U133 plus 2.0 chips. Un- factors that can play a role in trophoblast proliferation and/or supervised hierarchical cluster analysis resulted in a dendrogram differentiation. The expression of EGFR2 (also known as CD340 where each cell type formed a distinct cluster (Fig. 2A). As or ERBB2), LIFR, and, at low level, EGFR1 on EVT is confirmed, expected, DSC formed a separate branch from the other three and, moreover, a marked increase in expression of EGFR2 and cell types, whereas EVT were closer to JEG3 than to VT. LIFR, but not EGFR1 was observed after culture on fibronectin To evaluate the genomic differences that distinguish VT and (Fig. 1E) (2). VT expressed higher levels of EGFR1 and did not EVT, a volcano plot was generated based on the mean expres- express EGFR2 or LIFR. Culture on fibronectin did not change the sion value of an individual probe’s fold change and the P value expression on VT (SI Appendix,Fig.S3A). In addition, trophoblast associated with reproducibility of these changes between the VT isolates were cultured on fibronectin for 1 or 2 days and stained for and EVT (Fig. 2B). This identified 2,252 probes up-regulated

7220 | www.pnas.org/cgi/doi/10.1073/pnas.1507977112 Tilburgs et al. Downloaded by guest on October 1, 2021 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 al- though distinct probes were also identified (SI Appendix,Fig.S4B 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 family of cell ad- hesion 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 de- velopment of EVT (28). In addition to the integrin subunits that are differentially expressed (ITGA5 on EVT and ITGB5 and ITGB8 on VT), the α-andβ-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-reg- ulation of HLA-G. Integrin αVβ5 and αVβ8,bothvitronectin receptors, are uniquely expressed on VT. Three integrins are expressed by both EVT and VT: α6β4 and α6β1 (both laminin receptors) and αVβ1 (a vitronectin receptor). FACS and micro- array 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 differ- ences that reflect the transcriptional differences between EVT and VT, a Gene Set Enrichment Analysis (GSEA) was per- formed. 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- Fig. 2. Expression profiling of EVT and VT. (A) Unsupervised hierarchical cluster surface molecule CD276 (B7-H3) and cytotoxic and regulatory analysis results in a dendrogram where each distinct celltype(VT,EVT,JEG-3,and T-cell molecule (CRTAM), which can all directly influence lym- DSC) forms a separate cluster. Volcano plots were generated based on the mean phocyte binding and are potent inhibitors of lymphocyte activa- expression value of an individual probe’s fold change and the P value associated tion (SI Appendix,Fig.S7). EBI3 is known to dimerize with two with reproducibility of these changes between (B) VT and EVT and (C)EVTand α-chains, IL-12(p28) and IL-12(p35), to form the immune-sup- JEG-3. The unique gene signatures based on a more than fourfold difference for pressive cytokines IL-27 and IL-35 (30). However, neither of these VT (340 probes, blue dots) and EVT (328 probes, red dots) are highlighted. 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 INFLAMMATION in VT and 1,953 probes up-regulated in EVT with a twofold sets enriched in VT were less significant (increased false discovery IMMUNOLOGY AND difference and indicated that VT and EVT are very distinct cell rate) and more diverse in function (SI Appendix, Table S3). types. To generate more stringent gene signatures for VT and EVT, genes were selected based on fourfold differential expres- Coculture of EVT with Sample Matched Maternal Leukocytes. To eval- sion present in all seven patient pairs. This identified 340 probes uate the implications of the striking immune-activating and -reg- (encoding 221 unique genes after exclusion of duplicate genes and ulating potential of EVT demonstrated in the GSEA, VT- and uncharacterized loci) that were up-regulated in VT (Fig. 2B, blue EVT-enriched cocultures with all major maternal leukocyte subsets dots) whereas 328 probes (encoding 210 unique genes) were up- (dNK, dMɸ,CD4+,andCD8+ dT cells) were established. For this regulated in EVT (Fig. 2B, red dots). The annotated probe lists sample, matched trophoblast and decidual leukocytes or trophoblasts

Tilburgs et al. PNAS | June 9, 2015 | vol. 112 | no. 23 | 7221 Downloaded by guest on October 1, 2021 and peripheral blood leukocytes from unrelated nonpregnant donors not CD4+CD25HI Treg) were added to EVT, no induction of were used. The FACS sorting strategy for all cell types can be found CD4+CD25HIFOXP3+ Treg was observed (SI Appendix, Fig. in SI Appendix,Fig.S8. Microscopy images revealed that EVT adhere S10B). In contrast, when both total CD4+ pT and purified CD4+ to fibronectin with a distinct, large, spread-out morphology and in- CD25HI pT were cocultured with EVT, a marked increase in CD4+ teract with multiple other EVT. Furthermore, HLA-G+ EVT with CD25HIFOXP3+ Treg compared with CD4+ or CD4+CD25HI pT various sizes and morphology are found in the cell cultures whereas cultured alone was seen (SI Appendix, Fig. S10 A and C). Thus, VT are homogenous, small, rounded cells that do not adhere to EVT specifically increased FOXP3 expression in CD4+CD25HI fibronectin (SI Appendix,Fig.S9A). Light microscopy also revealed cells as opposed to a de novo induction of FOXP3 in CD4+ clustering of NK, CD4+,andCD8+ T cells around the large EVT. CD25− T cells. CD4+ pT or CD4+CD25HI pT were labeled with Multiple NK or T cells can interact with one EVT, either at the cell CFSE and incubated with or without EVT, VT, or anti-CD3/ body or at filopodia-like structures that spread out from the EVT CD28 beads. Only the pT cells incubated with anti-CD3/CD28 cell body (SI Appendix,Fig.S9B). No signs of cytolysis of EVT were beads showed proliferation at day 3, demonstrating that the observed in any of the trophoblast–leukocyte cocultures. increase in FOXP3 expression is not due to increased pro- liferation of these cells (SI Appendix,Fig.S10D). Furthermore, HI EVT Increase the Fraction of CD4+CD25HIFOXP3+CD45RA+ Treg. Treg when purified pCD4+CD25 cells were cocultured with EVT, are major regulators of immune responses at the maternal–fetal a significant increase of CD4+CD25HIFOXP3+ CD45RA+ interface (18, 31). To assess the influence of EVT on Treg, Treg, previously described to be resting Treg (32), was observed CD4+ dT and peripheral blood CD4+ T cells (CD4+ pT) were in the EVT but not in the VT cultures (Fig. 3 D and E). Thus, cultured alone and together with VT and EVT for 3 days. T cells EVT but not VT directly influence the generation of CD4+ were harvested and analyzed for the presence of CD4+CD25HI CD25HIFOXP3+CD45RA+ resting Treg. FOXP3+ Treg. Coculture of CD4+ dT and CD4+ pT with EVT significantly increased the percentage of CD4+CD25HIFOXP3+ EVT Do Not Elicit Specific Cytokine Responses by Maternal dNK, dMϕ, Treg. In addition, EVT significantly increased the expression or dT Cells. Cell culture supernatants were harvested from EVT- level (mean fluorescence intensity, or MFI) of FOXP3 within the enriched and leukocyte cocultures at day 1 (NK) and day 3 (Mɸ CD4+CD25HIFOXP3+ Treg after coculture with CD4+ pT and T cells), and cytokines were analyzed by using a multiplex (Fig. 3 A–C). The FOXP3 level in CD4+CD25HI dT was al- cytokine assay. As a negative control, all cell types were cultured ready very high and did not further increase by coculture with alone and as positive controls with 721.221 (221) MHC class I EVT. When CD4+CD25HI-depleted CD4+ pT (containing both negative target cells, with 721.221/HLA-G (221.HLA-G) or with naive CD4+CD25− and activated CD4+CD25DIM T cells but 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 proin- flammatory cytokines granulocyte monocyte–colony-stimulating A CD4+ pT CD4+ pT +EVT CD4+ pT +VT CD4+ pT factor (GM-CSF), IFN-gamma (IFNγ), and tumor necrosis fac- 2.0% 3.5% 2.5% 0.1% tor-alpha (TNFα) compared with dNK (Fig. 4 A–C). Recip- rocally, upon stimulation with 221 cells (but not with PMA),

IgG dNK secreted significantly higher levels of vascular endothelial FOXP3 growth factor (VEGF) and interleukin-6 (IL-6) than pNK (Fig. 4 D and E), confirming previous studies (6, 13, 22). Interestingly, CD25 expression of HLA-G on 221 cells reduced IL-6 secretion in BC dNK. Thus, HLA-G expression may play a direct role in sup- pression 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 D CD25HI pT CD25HI pT +EVT CD25HI pT +VT CD25HI pT (or in pNK) (Fig. 4 A–F). Thus, PMA, 221 cells, and 221/HLA-G

15.2% 3.0% 18.7% 5.6% 15.1% 4.1% 0% 0% 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 – IgG and EVT dNK interactions, primary EVT need to be included in FOXP3 further studies. Secretion of IL-8 (neutrophil chemotactic factor) CD45RA and galectin-1 (involved in suppression of activated T cells) was EF 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 re- sponsible 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 Fig. 3. Coculture with EVT increases the percentage of CD4 FOXP3 Treg. differences were found in cytokine responses between dNK or (A) Representative FACS plots of CD25 and FOXP3 expression of CD4+ pT in KIR2DS1+ KIR2DS1− the absence of stimulation or after coculture with VT- or EVT-enriched pNK from and individuals toward any of + + the target cells, including EVT. preparations. (B) The percentage of FOXP3 cells within total CD4 pT and ϕ dT and (C) MFI of FOXP3 expression within CD4+CD25HIFOXP3+ pT and dT Macrophages. As demonstrated previously, dM constitutively se- cocultured with or without VT- or EVT-enriched preparations. (D) Repre- crete both the pro- and anti-inflammatory cytokines IL-10, IL-1β, sentative FACS plots of CD45RA and FOXP3 within CD4+CD25hi pT cocul- TNFα, and IL-6 (19). However, in contrast to stimulation with tured with or without VT- or EVT-enriched preparations. (E) The percentage lipopolysaccharide (LPS) or LPS/IFN-γ (19), stimulation of dMϕ of FOXP3+CD45RA− and (F) FOXP3+CD45RA+ cells within each group. or monocytes VT- or EVT-enriched preparations did not change

7222 | www.pnas.org/cgi/doi/10.1073/pnas.1507977112 Tilburgs et al. Downloaded by guest on October 1, 2021 - molecules related to distinct functions (28, 35). In that context, the ABpNK present preparation may represent EVT at an intermediate level of dNK 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 C D 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 conver- sion of CD4+CD25HI FOXP3− cells into FOXP3+ Treg. In ad- dition, EVT, but not VT, significantly increased the proportion of CD4+CD25HIFOXP3+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 responsi- EFble 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- G H 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. Fig. 4. Cytokine secretion profiles of pNK and dNK during coculture with VT Previous studies using different isolation protocols may have and EVT. pNK (blue) and dNK (red) were incubated alone or with PMA, 221, selected trophoblasts with distinct characteristics (37). Further 221/HLA-G, and VT- or EVT-enriched preparations for 18 h. Cell culture su- effort needs to be made to characterize distinct VT and EVT types pernatants were analyzed for (A)GM-CSF,(B)IFNγ,(C)TNFα,(D) VEGF, (E)IL-6, present in placental tissue. The gene set enrichment analysis (F)IL-17A,(G) IL-8, and (H) galectin-1. VT- or EVT-enriched preparations cul- revealed a striking immune-activating potential for EVT and tured alone secreted IL-6 and IL-8 (E and G, depicted by gray dots), but did not identified genes able to directly modulate trophoblast–lymphocyte secrete any of the other cytokines. 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 the secretion of any of these cytokines (SI Appendix,Fig.S11). IL- (B7-H3) and CRTAM that can directly influence NK and T-cell 12(p70), a potent T-cell–stimulating cytokine, and IL-33, a mem- binding to EVT. Furthermore, the EVT gene signature revealed ber of the IL-1 family recently implicated in enhancing proliferation that EVT express a variety of cytokine receptors that may of primary trophoblasts (33) and promotion of Treg survival (34), render them responsive to cytokines and chemokines secreted were not detected in any of the dMϕ or monocyte cultures. α + + by leukocytes such as IL-10, IL-2, IL-1, MIP-1 , RANTES, T cells. CD4 and CD8 dT and pT were incubated with or without MCP-3, GM-CSF, M-CSF, and G-CSF. anti-CD3/CD28 beads and VT- or EVT-enriched preparations for + + Previous studies have suggested that EVT are derived from 3 days. Although both CD4 and CD8 dT and pT had a profound VT at the end tips of the villous tree. In this study, 4,205 probes IFNγ response to anti-CD3/CD28 beads, none of the T cells se- γ SI Appendix that are differentially expressed between EVT and VT were iden- creted IFN in response to VT or EVT ( ,Fig.S12). tified (2,252 probes are overrepresented in EVT and 1,953 in VT with a twofold difference). Furthermore, the GSEA and the unique Discussion VT and EVT gene signatures revealed major differences in cellular EVT are found in at least four different locations in the pregnant functions and expression of transcription factors. These large dif- uterus, i.e., as the cells at the tips of anchoring villi in cell col- ferences may indicate that VT and EVT are derived from a distinct umns (columnar EVT), as interstitial EVT within the decidua in cellular origin. Alternatively, EVT precursor cells or trophoblast contact with maternal leukocytes, as endovascular EVT within stem cells at the end tips of the villous tissue may give rise to fully

the uterine spiral arteries in contact with the vascular endothe- differentiated EVT. The proliferation and/or outgrowth of EVT INFLAMMATION lium, and as cells in chorionic tissue directly adjacent to the maternal observed by BrDU incorporation in placental explant cultures (7) IMMUNOLOGY AND decidual or parietal surface (chorionic EVT). In interpreting the may represent a growth burst derived from specialized precursor present data, it is important to note the origin of the EVT studied cells that differentiate into EVT or proliferation by HLA-G+ here. These cells were obtained from first-trimester villous tissue cells at the villous tip. Alternatively, VT may include distinct separated from the decidua. They largely represent cells obtained cell types (e.g., EGFR1+ and TACSTDC2+) of which some can from cell columns at the tips of anchoring villi. Interstitial EVT and and others cannot differentiate into HLA-G–expressing cells. endovascular EVT would represent a very small population—too Furthermore, cell culture conditions used here may lack additional small to be isolated under the present circumstances. Some data growth factors, integrin ligands, or oxygen/CO2 levels required for suggest that these EVT are all distinct in expression of different trophoblast differentiation.

Tilburgs et al. PNAS | June 9, 2015 | vol. 112 | no. 23 | 7223 Downloaded by guest on October 1, 2021 Because of the difficulties in obtaining human HLA-G+ EVT, Hospital (Boston, MA). All of the human tissue used for this research was de- many studies have attempted to generate human HLA-G+ EVT identified, discarded clinical material. The Committee on the Use of Human lines. However, careful assessment of the cell lines and their Subjects (the Harvard Institutional Review Board) determined that this use of MHC expression demonstrates that these cell lines do not express placental and decidual material is not human subjects research. Decidual and a similar MHC profile compared with EVT (38). The closest villous tissues were macroscopically identified and separated. Trophoblasts were model cell line for EVT and its atypical HLA-C, HLA-E, and isolated as described previously (26, 39). In short, villous tissue was scraped from HLA-G expression remains the choriocarcinoma cell line JEG-3. the basal membrane and digested for 8 min at 37 °C with trypsin (2 g/L) and The transcriptional profiles of JEG-3 differed from EVT by 1,475 EDTA (0.2 g/L). Trypsin was quenched with F12 medium with 1.0 mL/1 mL probe sets, many representing key functions of EVT, e.g., cyto- newborn calf serum and penicillin/streptomycin(Gibco)andfilteredovera kine production, growth factor receptors, ECM components and gauze mesh. Filtrate was layered on Ficoll (GE Healthcare) for density gradient enzymes, and MHC regulatory molecules. In this study, robust centrifugation (20 min at 800 × g) and thereafter incubated for 20 min at 37 °C and highly purified VT and EVT preparations were obtained and in a culture dish for removal of macrophages. Nonadherent cells were collected used to establish cocultures with decidual leukocytes. Most in- and directly stained for flow cytometric analysis or FACS sorting. terestingly, in contrast to previous studies that used surrogate Decidual tissue was washed, minced, and digested with 0.1 mg/mL col- HLA-G+ cell lines, HLA-G+ EVT did not elicit a profound cy- lagenase IV and 0.01 mg/mL DNase I (Sigma) shaking in a water bath for 1 h at tokine response by dNK, pNK, dMϕ, monocytes, and CD4+ or 37 °C. Released lymphocytes were filtered through 100-, 70-, and 40-μmsieves CD8+ dT or pT, even though the distinct cytokine secretion (BD Labware). Lymphocytes were dissolved in 20 mL 1.023g/mL Percoll (GE profiles of dNK and pNK in response to stimulation either by 221 Healthcare) and layered on 10 mL 1.080g/mL and 12.5 mL 1.053g/mL Percoll cells or 221/HLA-G target cells or by PMA was confirmed. for density gradient centrifugation (30 min at 800 × g). Lymphocytes were Thus, careful and systematic validation of EVT–leukocyte in- isolated from the 1.080 to 1.053 g/mL interface and dMϕ from the 1.053 to teractions needs to be carried out by using primary HLA-G+ 1.023 g/mL interface. Cells were washed and directly stained for flow cyto- EVT to understand the unique contribution of EVT to the de- metric analysis or FACS sort (SI Appendix, Fig. S8). pNK, pT, and monocytes cidual immune response in human pregnancy. The surrogate cell from leucopacks were isolated with RosetteSep (StemCell Technologies). For lines presently available are not adequate for this purpose. all leukocyte types, >95% purity was obtained. Studies on the role of primary EVT in maternal–fetal tolerance Cocultures, RNA isolation, microarray hybridization and analysis, flow will be crucial to understanding the development of pregnancy cytometry, cytokine analysis, and imaging and statistics used are described in complications such as preterm delivery and preeclampsia. SI Appendix. Materials and Methods ACKNOWLEDGMENTS. We thank Patricia Rogers and Joyce Lavecchio for help with cell sorting; Jennifer Couget for help with microarray experiments; Discarded human placental and decidual material was obtained from women and all past and current laboratory members for their helpful discussions. – undergoing elective pregnancy termination at 6 12 wk at a local reproductive This work was supported by National Institutes of Health Grant AI053330. health clinic. Peripheral blood leukocytes were isolated from discarded leuko- Â.C.C. was supported by the Portuguese Foundation for Science and Technology packs from healthy volunteer blood donors from the Massachusetts General (FCT) (SFRH/BD/33885/2009).

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