Human HLA-G+ extravillous trophoblasts: 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 trophoblast 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 cytotrophoblasts and syncytiotrophoblasts, 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.