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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2002 Oct 4;99(21):13741–13746. doi: 10.1073/pnas.162491699

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 , Jack L Strominger *,¶,‖
PMCID: PMC129762  PMID: 12368486

Abstract

Invariant CD1d-restricted natural killer T (iNKT) cells comprise a small, but significant, immunoregulatory T cell subset. Here, the presence of these cells and their CD1d ligand at the human maternal–fetal interface was investigated. Immunohistochemical staining of human decidua revealed the expression of CD1d on both villous and extravillous trophoblasts, the fetal cells that invade the maternal decidua. Decidual iNKT cells comprised 0.48% of the decidual CD3+ T cell population, a frequency 10 times greater than that seen in peripheral blood. Interestingly, decidual CD4+ iNKT cells exhibited a striking Th1-like bias (IFN-γ production), whereas peripheral blood CD4+ iNKT clones exhibited a Th2-like bias (IL-4 production). Moreover, compared to their peripheral blood counterparts, decidual iNKT clones were strongly polarized toward granulocyte/macrophage colony-stimulating factor production. The demonstration of CD1d expression on fetal trophoblasts together with the differential pattern of cytokine expression by decidual iNKT cells suggests that maternal iNKT cell interactions with CD1d expressed on invading fetal cells may play an immunoregulatory role at the maternal–fetal interface.

Invariant CD1d-restricted natural killer T cells (hereafter designated as iNKT cells) appear to play an important immunoregulatory role in the immune system. iNKT cells possess invariant TCRα chains, in which a Vα24JαQ junction is formed without N or P additions, preferentially paired with noninvariant Vβ11 TCRβ chains (1). An endogenous natural activating ligand has yet to be identified, although the marine sponge-derived glycolipid α-galactosylceramide (αGalCer), when bound and presented by CD1d, activates iNKT cells (2, 3). Activated iNKT cells are able to secrete a wide variety of cytokines and this may, in part, explain their ability to exert their effects over a wide immunological spectrum, such as in the onset of diabetes in humans and in NOD mice (4–8), the clearance of certain tumors (9, 10), the immune response to certain viruses (11–13), and as mediators of anterior chamber acquired immune deviation (ACAID) in which specific tolerance is generated to antigens introduced into the anterior chamber of the eye (14, 15). Notably, iNKT cells also have been implicated in allograft survival (16–19).

During pregnancy, hemi-allogeneic fetal trophoblast cells migrate from the chorionic villi and invade the maternal decidual tissue, eventually invading the maternal spiral arteries. The mechanisms that regulate trophoblast invasion remain poorly defined. Extravillous trophoblasts do not express HLA-A or -B molecules (20), but they do express HLA-C (21) as well as HLA-G (22), a nonclassical MHC class I molecule whose function is unknown. In mice, active induction of maternal tolerance to trophoblast-expressed antigens has been observed in transgenic models (23) and is probably the result of multiple immunological mechanisms present at the maternal–fetal interface to prevent rejection of the fetus, such as the regulation of tryptophan catabolism by indoleamine 2,3-dioxygenase in decidual macrophages and the expression by fetal trophoblasts of complement regulatory proteins (24). Trophoblasts may themselves play an active immunological role in regulating the immune response to the fetal allograft because they express immunologically relevant molecules such as IL-10 (25, 26) and granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (27).

Here, we demonstrate in humans the expression of CD1d on both villous and invasive extravillous fetal trophoblasts. In addition, human decidual iNKT cells, present at a frequency ≈10 times that seen in peripheral blood, exhibited a marked Th1-like cytokine bias as well as a striking polarization toward GM-CSF production. These data suggest that decidual iNKT cell interactions with CD1d-expressing trophoblasts play a specific role at the maternal–fetal interface, possibly in the acceptance of the fetal allograft and/or in placental development.

Materials and Methods

Abs and Fluorescence-Activated Cell Sorter (FACS) Analysis.

The following Abs were used in FACS analysis and immunohistochemistry: 6B11 (an mAb specific for the invariant Vα24JαQ CDR3 loop (M.E., F.K.R., J.E.B., J.L.S., S.P.B., and S.B.W., unpublished results); anti-CD1d 42.1 (28); anti-Vα24 and anti-Vβ11 (Coulter); anti-CD3 used in proliferation assays, T3D; anti-CD4, anti-CD8, anti-CD16, anti-CD56, anti-CD57, anti-CD69, anti-CD161, anti-CD45RO, anti-CD45RA, anti-CD94, IgG isotype controls, anti-IFN-γ, anti-IL-4, anti-GM-CSF, anti-IL-10, and anti-IL-2 were all from BD Biosciences (Los Angeles); NOR3.2 (BioSource International, Camarillo, CA), and anti-c-erbB-2 (NCL-CB11; NovoCastra, Newcastle, U.K.). For FACS staining, cells were washed in staining buffer [PBS (pH 7.2) supplemented with 2% FCS and 0.1% sodium azide]. Cells were incubated with mAb on ice for 30 min, then washed twice with staining buffer.

Tissues and Cell Culture.

Peripheral blood mononuclear cells were obtained from leukocytes discarded from aphoresis or from peripheral blood obtained by venipuncture. First trimester decidua from patients undergoing elective abortion were collected at the New York University Medical Center, New York, and the Brigham and Women's Hospital, Boston. Decidual tissue was washed extensively in PBS supplemented with 50 μg/ml gentamicin before mincing with sterile scissors. Decidual lymphocytes were released by digesting the tissue with 0.1% collagenase and 0.05% DNase I (both from Sigma). Lymphocytes were purified by density gradient centrifugation (Ficoll-Hypaque; Amersham Pharmacia and Upjohn), and cells were allowed to adhere to 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 either Vα24+Vβ11+ or 6B11+Vα24+ T cells at 1 cell per well in 96-well round-bottom plates. Peripheral blood mononuclear cells (γ-irradiated; 5,000 rads) were used as feeders, and phytohemagglutinin (Remel, Lenexa, KS) was added to a final concentration of 1 μg/ml. Cells were cultured in RPMI medium 1640 supplemented with 15% human AB serum (Atlanta Biologicals, Norcross, GA), penicillin/streptomycin, 100 mM sodium pyruvate, 1% nonessential amino acids, 2 mM l-glutamine, and 2-mercaptoethanol (all from Life Technologies, Rockville, MD). IL-2 (National Cancer Institute–Frederick Cancer Research and Development Center, Frederick, MD) was added to a final concentration of 100 units/ml. In some cases, peripheral iNKT clones were grown in 20 units/ml IL-2 plus 15 ng/ml IL-7 (R & D Systems). No difference was observed in functional assays between the two growth conditions. Clones were restimulated with 50,000 γ-irradiated feeders and phytohemagglutinin (1 μg/ml) every 3–4 wk.

Functional Analyses.

For iNKT cell activation assays, 5 × 104 iNKT cells were plated in a 96-well round-bottom plate with either medium alone or with 5 × 104 stimulator cells that had been treated with 0.1 mg/ml mitomycin C for 1 h at 37°C. αGalCer (KRN7000, a gift from Kirin Brewery, Tokyo), resuspended in DMSO was added at a final concentration of 100 ng/ml. Either phytohemagglutinin (1 μg/ml) or plate-bound anti-CD3 (1 μg/ml) was used as a positive control. For stimulation with CD1d transfectants, IL-2 was included in the culture medium at a final concentration of 20 units/ml. C1R B-LCLs and HeLa cells transfected with CD1d have been described (29). Typically, proliferation and cytokine release assays were performed 3–4 wk after the last restimulation. Proliferation was measured in a 96-h assay in which 1 μCi [3H]thymidine (1 Ci = 37 GBq) was added during the last 18 h of incubation, followed by harvesting and scintillation counting. Cytokine levels were measured by collecting supernatants after 48 h of incubation and testing by ELISA. Abs for GM-CSF detection were from R & D Systems, and Abs for IFN-γ and IL-4 were from BioSource. Streptavidin-POD (Roche Diagnostics) was used for detection with TMB substrate (Pierce). Cytotoxicity assays were performed no later than 10 d after the last restimulation. Target cells were labeled with [35S]methionine and culturing iNKT effector cells with targets for 5 h at effector-to-target cell ratios of 50, 25, and 12.5 to 1. NaOH (1 N) was added to induce maximum release. When αGalCer was used, it was added to the target cells the night before the assay at 100 ng/ml final concentration. Percentage of specific lysis was calculated by the following formula: [(experimental cpm − spontaneous release cpm)/(maximum release cpm − spontaneous release cpm)] × 100. Percentage of specific lysis was always <25%. All measurements were performed in triplicate.

Immunohistochemistry.

Decidual tissue was identified macroscopically and washed extensively with PBS, pH 7.2. Decidual pieces were fixed overnight at 4°C in 4% paraformaldehyde, after which they were washed in PBS, dehydrated in ethanol followed by xylene, and embedded in paraffin. For staining, 6-μm sections were rehydrated, and they were either stained with Gill's hematoxyolin and eosin or they underwent antigen retrieval (Vector Laboratories). Slides were incubated in 3% H2O2 in methanol before staining to block endogenous peroxidase activity. NOR3.2 and anti-c-erbB-2 mAb binding was detected by using the streptavidin/peroxidase and visualized by using diaminobenzidine substrate (R.T.U. Vectastain kit; Vector Laboratories).

Results

CD1d Expression on Trophoblasts.

To determine whether CD1d was expressed in the human placenta, immunohistochemical staining on paraffin-embedded sections of 8-, 10-, 38-, and 39-wk-old human decidua was performed. CD1d expression detected by the CD1d-specific NOR3.2 mAb was clearly apparent in both villous and extravillous trophoblasts (Fig. 1). Extravillous trophoblast were identified by morphology in serial hematoxylin/eosin sections (Fig. 1A) and by the expression of the c-erbB-2 protein, which is expressed on extravillous but not villous trophoblast (Fig. 1E; ref. 30). Staining of serial sections with NOR3.2 demonstrated that CD1d was expressed on the c-erbB-2+ population (Fig. 1 E and F). CD1d also was present on villous trophoblasts (Fig. 1G) and on trophoblasts, which had been trapped in the fibrinoid deposits at the maternal–fetal interface (Fig. 1 D and H). Additional experiments will be needed to determine whether CD1d is expressed in all villous trophoblasts or only on those differentiating to the “intermediate” phenotype that comprises cell columns. A low level of syncytiotrophoblast staining with the NOR3.2 mAb was observed but it was judged to be inconclusive because a similar amount of syncytiotrophoblast staining was seen by using the isotype-matched control.

Figure 1.

Figure 1

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CD1d is expressed on human trophoblasts. Immunohistochemistry on paraformaldehyde-fixed decidual tissue (8 wk; 6-μm sections). (A) Hematoxylin/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.

Invariant NKT Cells in the Decidua.

To determine the frequency and phenotype of iNKT cells in the human decidua, FACS analysis of lymphocytes isolated from first trimester (7–10 wk) decidual tissue was performed. Vα24+Vβ11+ cells comprised 0.48 ± 0.05% (n = 4) of the CD3+ decidual lymphocytes, much higher than the 0.04 ± 0.02% (n = 4) frequency seen in human peripheral blood (Fig. 2 A and C). Similar results were obtained when anti-Vα24 mAb was used in conjunction with 6B11, a mAb raised against a cyclic peptide corresponding to the invariant CDR3α loop of iNKT cells (Fig. 2B). Thus, the decidual T cell population was greatly enriched in iNKT cells compared to the peripheral blood.

Figure 2.

Figure 2

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A high frequency of invariant NKT cells in the decidua. (A) Representative contour plots of CD3-gated lymphocytes stained with anti-Vα24 and anti-Vβ11 mAbs. (B) Contour plot of CD3-gated decidual lymphocytes stained with the anti-CDR3α mAb 6B11 and anti-Va24. (C) Summary of iNKT cell frequencies as a percentage of CD3+ T cells in the decidua and in the periphery. Mean percentages of iNKT frequencies are indicated by bars (C).

To verify that the 6B11+Vα24+ decidual T cells were indeed iNKT cells, these cells were cloned by single cell FACS sorting of 6B11+Vα24+ decidual T cells. PCR amplification and direct sequencing across the TCR CDR3α region indicated that the clones possessed characteristic TCR Vα24JαQ junctions without N/P additions. Because it is difficult to obtain T or iNKT cells in large numbers from decidual tissue for phenotypic analysis, a total of 32 iNKT clones were isolated from six different individuals and stained with a panel of mAbs. Importantly, 6B11+ iNKT cells were always Vα24+Vβ11+. Interestingly, although FACS analysis demonstrated that <40% of decidual iNKT cells were CD4+ (the remainder being CD4−), only CD4+ clones could be expanded. The reason for this is unclear, but it is possible that the decidual CD4− population may have some specific growth impairment or that additional factors may be required to expand these cells. Therefore, in this manuscript all decidual iNKT clones referenced are CD4+ clones. Like peripheral iNKT cells, decidual iNKT clones were all CD45RO+ and CD45RA−. Examination of the clones with a panel of mAb directed to NK markers revealed that the expression of CD161 (the human homolog of murine NK1.1) was quite variable and that although none of the decidual iNKT clones expressed the CD16 or CD94 molecules, many clones expressed low levels of CD57 and significant levels of CD56.

CD1d Reactivity of Decidual iNKT Cells.

Lymphocytes that populate the decidua are thought to have altered functional capabilities (31). Because the hallmark of iNKT cells is their ability to recognize CD1d, especially αGalCer bound by CD1d, the ability of decidual iNKT clones to recognize CD1d+ target cells was tested. In a 96-h standard proliferation assay, αGalCer, pulsed onto C1R cells transfected with CD1d, induced proliferation of decidual iNKT cells to a level similar to that of peripheral iNKT clones (Fig. 3A) and decidual iNKT clones efficiently killed αGalCer-loaded CD1d+ target cells (Fig. 3B), as previously shown for peripheral iNKT cells (3). In addition, decidual iNKT clones cocultured with either αGalCer-pulsed C1R/CD1d or αGalCer-pulsed HeLa/CD1d transfectants secreted both IFN-γ and IL-4, demonstrating that the decidual iNKT clones were CD1d-restricted (unpublished data). Interestingly, some clones exhibited low levels of reactivity to C1R/CD1d transfectants in the absence of αGalCer (clone 15-3 in Fig. 3A, and clones 21-29 and 21-10 in Fig. 3B), which presumably reflects reactivity toward endogenous ligands bound by CD1d.

Figure 3.

Figure 3

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Decidual iNKT cells are restricted by CD1d. iNKT clones were assayed for their ability to recognize CD1d-expressing cells. (A) Proliferation of peripheral (B2) and decidual (15-3 and 33-10) iNKT clones cocultured with mock-transfected C1Rneo cells and C1R cells transfected with CD1d, with and without αGalCer. Error bars represent the SD from the mean. Results are representative of 25 decidual iNKT clones. (B) Lysis by iNKT clones of C1Rneo and C1R/CD1d transfectants that had been pulsed with αGalCer or vehicle. Six representative decidual iNKT clones from two different patients are shown at an effector-to-target ratio of 50:1. Error bars represent the SD from the mean. Results are representative of 14 decidual iNKT clones.

The ability of decidual iNKT cells to secrete IL-10 was determined because IL-10 was demonstrated in the mouse to be an important regulatory cytokine produced by iNKT cells (15). Intracellular staining of decidual iNKT clones after stimulation with phorbol 12-myristate 13-acetate and ionomycin revealed little or no IL-10 production (unpublished data).

Decidual iNKT Cells Exhibit a Th1-Like Bias and a Polarization Toward GM-CSF Production.

Because iNKT cells appear to function, in part, by secreting a wide variety of cytokines, we asked whether there was a difference in the cytokine production of resident decidual and peripheral blood iNKT cells. Peripheral iNKT clones were FACS-sorted from four additional donors. Because all of the decidual clones were CD4+ iNKT cells, only CD4+ peripheral iNKT cells were selected for comparison. Three weeks after restimulation, quiescent clones were either incubated with medium alone or stimulated with either IL-2, plate-bound anti-CD3, or αGalCer-loaded C1R/CD1d transfectants. Cell culture supernatants were harvested after 48 h and tested by ELISA for GM-CSF, IFN-γ, and IL-4 production.

Surprisingly, decidual iNKT clones exhibited a striking bias toward IFN-γ secretion as opposed to IL-4 (Fig. 4). Peripheral iNKT clones, in contrast, were biased toward IL-4 production. Although some peripheral clones appeared predisposed to secrete IFN-γ, and some decidual clones exhibited little if any bias, there was a striking difference when the two populations were compared as a whole. The IFN-γ/IL-4 difference between the peripheral and decidual populations was detectable even in IL-2-stimulated clones, although the absolute level of cytokine secretion from these clones was very low. Similar results were obtained whether the clones were stimulated with anti-CD3 or with α-GalCer-loaded C1R/CD1d transfectants (Fig. 4). Interestingly, in both peripheral and decidual iNKT clones, αGalCer stimulation resulted in a more pronounced Th2-like bias than did anti-CD3 stimulation—i.e., the Th2-like bias in peripheral iNKT clones was enhanced by using αGalCer, whereas the Th1-like bias in the decidual iNKT clones was diminished (Fig. 4).

Figure 4.

Figure 4

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Stimulation of decidual iNKT clones reveals a Th1-like bias in cytokine secretion. Decidual and peripheral iNKT clones were stimulated with IL-2, with C1R/CD1d transfectants pulsed with αGalCer, or with anti-CD3. Supernatants were collected after 48 h and tested for IFN-γ and IL-4 by ELISA. Data are expressed as log ratios of the amount of secreted cytokines. Each bar represents one individual clone. Peripheral and decidual clones were derived from three and six different individuals, respectively.

Next, GM-CSF levels were assessed because GM-CSF was previously demonstrated to be a highly regulated iNKT cell cytokine by microarray gene expression profiling (7). When iNKT clones were stimulated with either αGalCer or with anti-CD3, GM-CSF production by the decidual clones was significantly greater than that by the peripheral clones (Fig. 5). The difference in mean GM-CSF production was greatest in anti-CD3-stimulated cells (decidual, 11,700 pg/ml; peripheral, 2,750 pg/ml, P < 0.001) and slightly less so in αGalCer-stimulated cells (decidual, 3,680 pg/ml; peripheral, 1,640 pg/ml, P < 0.001). Upon IL-2 stimulation, the peripheral iNKT clones produced minimally higher mean levels of GM-CSF than the decidual iNKT clones, 140 and 55 pg/ml, respectively (Fig. 5). The ELISA data were confirmed by comparing peripheral and decidual CD4+ iNKT clone cytokine transcript levels by using ribonuclease protection assays. In accordance with the ELISA, decidual iNKT clones produced higher relative levels of IFN-γ and GM-CSF transcripts than peripheral iNKT clones, whereas peripheral iNKT clones exhibited higher levels of IL-4 transcripts (unpublished data).

Figure 5.

Figure 5

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Decidual iNKT cells produce much higher amounts of GM-CSF than their peripheral counterparts. GM-CSF was measured by ELISA from decidual and peripheral iNKT clones cultured with IL-2, or stimulated with anti-CD3 or with αGalCer-pulsed C1R/CD1d transfectants. ○, Individual clones (decidual, n = 24; peripheral n = 18), and the geometric mean of the GM-CSF level for each condition is denoted by a bar. The geometric means of GM-CSF levels for decidual clones were: IL-2-stimulated, 55 pg/ml; αGalCer-stimulated, 3,680 pg/ml; anti-CD3-stimulated 11,700 pg/ml. Geometric means of GM-CSF levels for peripheral clones were: IL-2-stimulated, 140 pg/ml; αGalCer-stimulated, 1,640 pg/ml; anti-CD3-stimulated, 2,750 pg/ml.

Discussion

Numerous reports have demonstrated that iNKT cells are key regulators of autoimmunity and immune tolerance (4–8, 14, 15, 32, 33) and may be important in graft survival (16–19). Active tolerance induction occurs at the maternal–fetal interface, suggesting it is one site in which iNKT cell–CD1d interactions may occur. In pregnant mice, αGalCer efficiently induces abortion via an iNKT-dependent mechanism (34), possibly as the result of overstimulation of a normal physiological mechanism that may be analogous to the systemic release of cytokines induced by superantigens. Evaluating the possible functions of iNKT cells at the maternal–fetal interface is difficult because CD1d expression has not been evaluated. Therefore, to investigate whether maternal iNKT cell–CD1d interactions may play a role in normal physiological processes at the human maternal–fetal interface, the presence of CD1d was examined and decidual iNKT cells were compared to their peripheral blood counterparts.

Interestingly, CD1d was expressed on trophoblasts, the fetal cells that invade the maternal tissue. CD1d expression has previously been reported in myeloid lineage cells, B cells, and activated T cells (28) as well as on intestinal and reproductive tract epithelia (35, 36). Immunohistochemical staining demonstrated CD1d expression on both villous and extravillous trophoblasts [as judged by costaining of serial sections for the epidermal growth factor-like c-erbB-2, an extravillous trophoblast marker (30)], but not syncytiotrophoblast. These data are consistent with previous work demonstrating CD1d transcripts in trophoblasts and choriocarcinoma cell lines, although full-length transcripts were never identified (37). Attempts to detect CD1d on JEG-3 choriocarcinoma cells by FACS and to up-regulate its expression by using IFN-γ were unsuccessful (J.E.B., unpublished data). Human trophoblasts do not express HLA-A and HLA-B molecules (20), presumably to escape allo-recognition by the maternal immune system. Upon their differentiation to the invasive extravillous phenotype, trophoblasts begin to express the nonclassical molecule HLA-G (22, 38), whose function is unknown. Thus, it is probable that CD1d and HLA-G are coexpressed on the invasive extravillous trophoblast subset, though this has not been formally demonstrated.

The frequency of iNKT cells observed in decidua was considerably higher than that found in peripheral blood. A similar frequency of decidual Vα24+CD161+ cells, presumably iNKT cells, was reported recently (50), but these cells were not further characterized. In the present study, FACS staining of freshly isolated decidual lymphocytes revealed the presence of both CD4+ and CD4− iNKT cells, yet only CD4+ cells were recovered after cloning. Whether this reflects a growth impairment on the part of CD4−CD8− decidual iNKT cells is unknown; although we have observed that decidual NK cells demonstrate diminished proliferative and cytolytic capacities compared to peripheral blood NK cells (J.E.B. and L.A.K., unpublished data). Examination of decidual iNKT cell phenotypic markers revealed few differences from peripheral blood iNKT cells. One significant difference, however, was that all decidual iNKT clones were CD94−, whereas CD94 is expressed on ≈50% of peripheral blood iNKT cells. This difference probably reflects the fact that only CD4+ clones were examined here, because a recent report using αGalCer-loaded tetramers suggests that CD94 is preferentially expressed on CD4− clones (39).

Because previous reports suggest that iNKT cells may function, in part, by differential cytokine expression, the expression profile of decidual CD4+ iNKT clones and peripheral CD4+ iNKT clones was compared. Intracellular staining for cytokine expression revealed the production of GM-CSF, IFN-γ, and IL-4. When expression of these cytokines was compared between peripheral and decidual iNKT clones, two striking differences were observed. First, GM-CSF production, measured by ELISA, was much higher in the decidual iNKT clones than in the peripheral iNKT clones. These data were corroborated by ribonuclease protection assay analysis, which demonstrated higher GM-CSF transcript levels in decidual iNKT cells than in peripheral iNKT cells. GM-CSF is a pleiotropic cytokine, which has a demonstrated ability to affect pregnancy outcome. GM-CSF−/− mice show selective impairment in their ability to reproduce (40), whereas administration of GM-CSF to mice in the abortion-prone CBA/J × DBA/2 mating combination results in a dramatic decrease in fetal resorption (41). Interestingly, mice lacking the GM-CSF receptor common β chain possess fewer iNKT cells than control mice as well as a defect in iNKT maturation (42). GM-CSF receptor is expressed on extravillous trophoblast cells, the very cells on which CD1d is expressed (27), suggesting that GM-CSF production in response to iNKT–trophoblast interaction may have implications for the function or fate of the trophoblast. Alternatively, GM-CSF production may play a role in the maturation of myeloid lineage cells in the decidua. Decidual macrophages, in turn, have the capability to suppress allogeneic T cell responses (43, 44).

A dramatic difference between decidual and peripheral iNKT clones in IFN-γ and IL-4 production also was observed. Decidual iNKT clones exhibited a clear bias toward IFN-γ secretion vs. IL-4 secretion. In contrast, peripheral blood CD4+ iNKT cells appeared to be biased toward IL-4 secretion in agreement with a previous report (45). These observations are consistent with RT-PCR results from mouse decidual iNKT cells (34), suggesting that a similar skewing toward Th1-like cytokines occurs in both species. Curiously, IFN-γ, together with tumor necrosis factor-α, mediates αGalCer-induced abortion in the mouse (34). Why would decidual iNKT cells possess a phenotype (Th1-like) associated with abortion induction? One possible explanation is that the abortions observed after αGalCer administration results from a systemic overproduction of these cytokines by iNKT cells in the periphery (as well as an overproduction at the maternal–fetal interface). Indeed, direct injection of IFN-γ or tumor necrosis factor-α effectively induces abortions in mice (41, 46). Conversely, IFN-γ appears to be necessary for the proper development of the decidua (47). Thus, any severe disruption to the tightly regulated balance of IFN-γ and/or other cytokines may be deleterious to the maintenance of pregnancy.

Qualitative and quantitative differences in the cytokines secreted by iNKT cells have been reported in both prostate cancer and type I diabetes (6, 7, 48). Recently, phenotypic differences between the CD4+ and CD4− iNKT cell subsets were described by two groups (39, 49). Whereas CD4− iNKT cells produce primarily Th1-type cytokines such as IFN-γ and tumor necrosis factor-α, CD4+ iNKT cells were reported to produce both Th1-type and Th2-type cytokines. The present results suggest that within the CD4+ iNKT cell subset, there may be differences in decidual and peripheral blood Th1/Th2 cytokine phenotypes. A shift of this balance in a manner similar to classical T helper subsets may account for the ability of iNKT cells to function in a variety of contexts.

Acknowledgments

We thank Ilaria Potolicchio, Hiroyuki Nishimura, and members of the Strominger laboratory for helpful discussion, Drs. Rapin Osathanondh and Dan Schust for supplying decidual samples, Maris Handley and Herb Levine for assistance in flow cytometry, and Dr. Raymond Genest for advice. We also thank Kirin Brewery for providing the KRN7000 (α-galactosylceramide). This research was supported by National Institutes of Health Grants CA-47554 and AI-50207 (to J.L.S.).

Abbreviations

iNKT cell

invariant natural killer T cell

αGalCer

α-galactosylceramide

GM-CSF

granulocyte/macrophage colony-stimulating factor

FACS

fluorescence-activated cell sorter

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