Decidual NK Cells Alter In Vitro First Trimester Extravillous Cytotrophoblast Migration: A Role for IFN- 1 Yuxiang Hu,* ¶ Jan P. Dutz, †¶ Colin D. MacCalman,* ¶ Paul Yong, †§¶ Rusung Tan, ‡¶ and Peter von Dadelszen 2 * ¶ Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi. The Journal of Immunology, 2006, 177: 8522– 8530. T he successful and tightly regulated interaction between the maternal immune system and fetal tissue is a prereq- uisite for normal pregnancy outcomes. Insufficient acqui- escence by the maternal immune system may lead to the inade- quate placentation of recurrent pregnancy loss, intrauterine growth restriction, and preeclampsia (1). Overzealous fetal invasion may lead to morbid adherence of the placenta, placenta accreta, increta, and percreta (2). It has been postulated that maternal decidual NK (dNK) 3 cells play an important role in the immune regulation of trophoblast invasion. There is considerable evidence to support that view (3). NK cells dominate during the early phase of gestation when pla- cental trophoblast cells invade into decidua (4), and are found par- ticularly around infiltrating trophoblast. This activity suggests a role in regulating the degree of invasion and differentiation of the trophoblast (5). Preeclampsia, and the related fetal growth restric- tion, are interpreted as consequences of impaired trophoblast in- vasion (1, 6). Severe reduction of trophoblast cells, in combination with a strong increase of maternal lymphocytes and dNK cells, is found in intrauterine growth restriction (7). Extravillous cytotrophoblast (EVT) express novel HLA-Ib pro- teins, HLA-G, and HLA-E (8, 9). These molecules have special roles in protecting trophoblast cells from maternal immune cells, especially dNK cells. In vitro studies have shown that the cytotoxic reactivity of dNK cells against HLA-G expression target cells of various origins was inhibited (10). HLA-E is a major ligand for the NK inhibitory receptor CD94/NKG2A (11), which mediates inhi- bition of killing of dNK cells (9). In the mouse, IL-15 was essential for the support of NK cell differentiation in the decidualizing uterus (12). dNK maintained with IL-15 in vitro have no cytotox- icity against trophoblast (13). This result suggests that, in normal pregnancy, dNK are not cytotoxic against cytotrophoblast. During implantation, cytotrophoblast leave the tips of anchoring villi and become EVT. Initially, the EVT form cell columns, then invade and migrate from the columns into the maternal tissue, the decidua. The EVT at the villus tip are  6  4 integrin positive, the EVT in the columns are  5  1 integrin positive, and the invading EVT are  1  1 integrin positive (14, 15). A well-established experimental model of placentation involves the use of chorionic villi from first trimester terminations of preg- nancy cultured as tissue explants on either collagen (two-dimen- sional model) (16, 17) or Matrigel (three-dimensional model) (18). In the two-dimensional model, the integrin  1 and  5 changes of invading EVT are not noted (16), whereas they are with the Ma- trigel model (18). IFN-, a cytokine secreted by both activated T lymphocytes and NK, is best known for its immunological functions. It has been used to treat viral infections (19, 20). However, the use of IFN- in pregnancy might exert a deleterious effect, such as increased miscarriage (21). IFN- halts trophoblast migration in explant cul- ture (22, 23), as does TNF-. Others have reported that the fol- lowing factors also contribute trophoblast migration inhibition: TGF- 1 , TGF- 2 , IL-10, and tissue inhibitor of metalloproteases (TIMP)-1 (17, 22, 24 –29). The role of dNK in controlling EVT differentiation and invasion has not been fully determined in a human model of placentation. *Department of Obstetrics and Gynaecology, † Department of Medicine, ‡ Department of Pathology and Laboratory Medicine, and § Department of Medical Genetics, Uni- versity of British Columbia, Vancouver, British Columbia, Canada; and ¶ Child and Family Research Institute, Centre for Healthcare Innovation and Improvement, and Programs in Infection and Immunity and Reproductive Health, Vancouver, British Columbia, Canada Received for publication March 15, 2006. Accepted for publication September 19. 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was funded by a Pilot Grant from the Infection and Immunity Program, Child and Family Research Institute. Y.H. is funded by grants from the Canadian Institutes for Health Research, from whom P.v.D. receives salary support. C.D.M. and P.v.D. receive salary support from the Child and Family Research Institute. P.v.D. and R.T. receive salary support from the Michael Smith Foundation for Health Research. 2 Address correspondence and reprint requests to Dr. Peter von Dadelszen, Depart- ment of Obstetrics and Gynaecology, University of British Columbia, 2H30-4500 Oak Street, Vancouver, British Columbia V6H 3N1, Canada. E-mail address: pvd@cw.bc.ca 3 Abbreviations used in this paper: dNK, decidual NK; EVT, extravillous cytotro- phoblast; TIMP, tissue inhibitor of metalloprotease; MTS, 3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAI, plasmino- gen activator inhibitor; CM, dNK-derived conditioned medium; HF, hollow fiber; IQR, interquartile range; MMP, matrix metalloprotease; uPA, urokinase-type protease activator. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00