CD1d and invariant NKT cells at the human maternal–fetal interface Jonathan E. Boyson*, Basya Rybalov*, Louise A. Koopman*, Mark Exley†, Steven P. Balk†, Frederick K. Racke‡, Frederick Schatz§, Rachel Masch§, S. Brian Wilson¶, and Jack L. Strominger*¶ʈ *Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; †Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; ‡Department of Pathology, Johns Hopkins University, Baltimore, MD 21287; §Department of Obstetrics and Gynecology, New York University Medical Center, New York, NY 10016; and ¶Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115 Contributed by Jack L. Strominger, August 15, 2002 Invariant CD1d-restricted natural killer T (iNKT) cells comprise a selves play an active immunological role in regulating the small, but significant, immunoregulatory T cell subset. Here, the immune response to the fetal allograft because they express presence of these cells and their CD1d ligand at the human immunologically relevant molecules such as IL-10 (25, 26) and maternal–fetal interface was investigated. Immunohistochemical granulocyte͞macrophage colony-stimulating factor (GM-CSF) staining of human decidua revealed the expression of CD1d on receptor (27). both villous and extravillous trophoblasts, the fetal cells that Here, we demonstrate in humans the expression of CD1d on invade the maternal decidua. Decidual iNKT cells comprised 0.48% both villous and invasive extravillous fetal trophoblasts. In of the decidual CD3؉ T cell population, a frequency 10 times addition, human decidual iNKT cells, present at a frequency Ϸ10 greater than that seen in peripheral blood. Interestingly, decidual times that seen in peripheral blood, exhibited a marked Th1-like CD4؉ iNKT cells exhibited a striking Th1-like bias (IFN-␥ produc- cytokine bias as well as a striking polarization toward GM-CSF -tion), whereas peripheral blood CD4؉ iNKT clones exhibited a production. These data suggest that decidual iNKT cell inter Th2-like bias (IL-4 production). Moreover, compared to their pe- actions with CD1d-expressing trophoblasts play a specific role at ripheral blood counterparts, decidual iNKT clones were strongly the maternal–fetal interface, possibly in the acceptance of the polarized toward granulocyte͞macrophage colony-stimulating fetal allograft and͞or in placental development. factor production. The demonstration of CD1d expression on fetal trophoblasts together with the differential pattern of cytokine Materials and Methods expression by decidual iNKT cells suggests that maternal iNKT cell Abs and Fluorescence-Activated Cell Sorter (FACS) Analysis. The interactions with CD1d expressed on invading fetal cells may play following Abs were used in FACS analysis and immunohisto- an immunoregulatory role at the maternal–fetal interface. chemistry: 6B11 (an mAb specific for the invariant V␣24J␣Q CDR3 loop (M.E., F.K.R., J.E.B., J.L.S., S.P.B., and S.B.W., ␣ nvariant CD1d-restricted natural killer T cells (hereafter unpublished results); anti-CD1d 42.1 (28); anti-V 24 and anti- ␤ Idesignated as iNKT cells) appear to play an important immu- V 11 (Coulter); anti-CD3 used in proliferation assays, T3D; noregulatory role in the immune system. iNKT cells possess anti-CD4, anti-CD8, anti-CD16, anti-CD56, anti-CD57, anti- ␣ ␣ ␣ CD69, anti-CD161, anti-CD45RO, anti-CD45RA, anti-CD94, invariant TCR chains, in which a V 24J Q junction is formed ␥ without N or P additions, preferentially paired with noninvariant IgG isotype controls, anti-IFN- , anti-IL-4, anti-GM-CSF, anti- V␤11 TCR␤ chains (1). An endogenous natural activating ligand IL-10, and anti-IL-2 were all from BD Biosciences (Los Ange- has yet to be identified, although the marine sponge-derived les); NOR3.2 (BioSource International, Camarillo, CA), and glycolipid ␣-galactosylceramide (␣GalCer), when bound and anti-c-erbB-2 (NCL-CB11; NovoCastra, Newcastle, U.K.). For presented by CD1d, activates iNKT cells (2, 3). Activated iNKT FACS staining, cells were washed in staining buffer [PBS (pH cells are able to secrete a wide variety of cytokines and this may, 7.2) supplemented with 2% FCS and 0.1% sodium azide]. Cells in part, explain their ability to exert their effects over a wide were incubated with mAb on ice for 30 min, then washed twice immunological spectrum, such as in the onset of diabetes in with staining buffer. humans and in NOD mice (4–8), the clearance of certain tumors (9, 10), the immune response to certain viruses (11–13), and as Tissues and Cell Culture. Peripheral blood mononuclear cells were mediators of anterior chamber acquired immune deviation obtained from leukocytes discarded from aphoresis or from (ACAID) in which specific tolerance is generated to antigens peripheral blood obtained by venipuncture. First trimester de- introduced into the anterior chamber of the eye (14, 15). cidua from patients undergoing elective abortion were collected Notably, iNKT cells also have been implicated in allograft at the New York University Medical Center, New York, and the Brigham and Women’s Hospital, Boston. Decidual tissue was survival (16–19). ␮ ͞ During pregnancy, hemi-allogeneic fetal trophoblast cells washed extensively in PBS supplemented with 50 g ml genta- migrate from the chorionic villi and invade the maternal decid- micin before mincing with sterile scissors. Decidual lymphocytes ual tissue, eventually invading the maternal spiral arteries. The were released by digesting the tissue with 0.1% collagenase and mechanisms that regulate trophoblast invasion remain poorly 0.05% DNase I (both from Sigma). Lymphocytes were purified defined. Extravillous trophoblasts do not express HLA-A or -B by density gradient centrifugation (Ficoll-Hypaque; Amersham molecules (20), but they do express HLA-C (21) as well as Pharmacia and Upjohn), and cells were allowed to adhere to HLA-G (22), a nonclassical MHC class I molecule whose tissue culture plates for 2–18 h at 37°C in a humidified 5% CO2, 37°C incubator. iNKT cell clones were derived by FACS sorting function is unknown. In mice, active induction of maternal ␣ ϩ ␤ ϩ ϩ ␣ ϩ tolerance to trophoblast-expressed antigens has been observed either V 24 V 11 or 6B11 V 24 T cells at 1 cell per well in transgenic models (23) and is probably the result of multiple in 96-well round-bottom plates. Peripheral blood mononuclear immunological mechanisms present at the maternal–fetal inter- face to prevent rejection of the fetus, such as the regulation of Abbreviations: iNKT cell, invariant natural killer T cell; ␣GalCer, ␣-galactosylceramide; tryptophan catabolism by indoleamine 2,3-dioxygenase in de- GM-CSF, granulocyte͞macrophage colony-stimulating factor; FACS, fluorescence- cidual macrophages and the expression by fetal trophoblasts of activated cell sorter. IMMUNOLOGY complement regulatory proteins (24). Trophoblasts may them- ʈTo whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.162491699 PNAS ͉ October 15, 2002 ͉ vol. 99 ͉ no. 21 ͉ 13741–13746 Fig. 1. CD1d is expressed on human trophoblasts. Immunohistochemistry on paraformaldehyde-fixed decidual tissue (8 wk; 6-␮m sections). (A) Hematoxylin͞ eosin staining of the maternal–fetal interface where trophoblasts (arrow) have migrated from chorionic villi toward the decidua. The eosinophilic (pink) material is fibrin deposited at the maternal–fetal interface. Staining of serial sections demonstrated that CD1d was expressed on the same cell population which expressed an extravillous trophoblast marker, c-erbB-2 (E and F). CD1d expression also was detected on villous trophoblasts (G) as well as on individual cells that had migrated from the villi (D and H). Isotype-matched control IgG mAb staining (B and C), CD1d staining by using NOR3.2 (D and F–H), and staining with anti-c-erbB-2 (E). DEC, decidua; IVS, intervillous space; VIL, villous. Arrows denote trophoblasts. cells (␥-irradiated; 5,000 rads) were used as feeders, and phy- after the last restimulation. Target cells were labeled with tohemagglutinin (Remel, Lenexa, KS) was added to a final [35S]methionine and culturing iNKT effector cells with targets concentration of 1 ␮g͞ml. Cells were cultured in RPMI medium for5hateffector-to-target cell ratios of 50, 25, and 12.5 to 1. 1640 supplemented with 15% human AB serum (Atlanta Bio- NaOH (1 N) was added to induce maximum release. When logicals, Norcross, GA), penicillin͞streptomycin, 100 mM so- ␣GalCer was used, it was added to the target cells the night dium pyruvate, 1% nonessential amino acids, 2 mM L-glutamine, before the assay at 100 ng͞ml final concentration. Percentage of and 2-mercaptoethanol (all from Life Technologies, Rockville, specific lysis was calculated by the following formula: [(experi- MD). IL-2 (National Cancer Institute–Frederick Cancer Re- mental cpm Ϫ spontaneous release cpm)͞(maximum release search and Development Center, Frederick, MD) was added to cpm Ϫ spontaneous release cpm)] ϫ 100. Percentage of specific a final concentration of 100 units͞ml. In some cases, peripheral lysis was always Ͻ25%. All measurements were performed in iNKT clones were grown in 20 units͞ml IL-2 plus 15 ng͞ml IL-7 triplicate. (R & D Systems). No difference was observed in functional assays between the two growth conditions. Clones were restim- Immunohistochemistry. Decidual tissue was identified macroscop- ulated with 50,000 ␥-irradiated feeders and phytohemagglutinin ically and washed extensively with PBS, pH 7.2. Decidual pieces (1 ␮g͞ml) every 3–4 wk. were fixed overnight at 4°C in 4% paraformaldehyde, after which they were washed in PBS, dehydrated in ethanol followed by Functional Analyses. For iNKT cell activation assays, 5 ϫ 104 iNKT xylene, and embedded in paraffin. For staining, 6-␮m sections cells were plated in a 96-well round-bottom plate with either were rehydrated, and they were either stained with Gill’s hema- medium alone or with 5 ϫ 104 stimulator cells that had been toxyolin and eosin or they underwent antigen retrieval (Vector ͞ ␣ treated with 0.1 mg ml mitomycin C for1hat37°C.