Natural killer cells promote immune tolerance byregulating inflammatory TH17 cells at the humanmaternal–fetal interfaceBinqing Fua, Xianchang Lib,c, Rui Suna, Xianhong Tongd, Bin Lingd, Zhigang Tiana,1, and Haiming Weia,1

aHefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Anhui 230027,China; Transplant Research Center, bBrigham and Women’s Hospital and cHarvard Medical School, Boston, MA 02115; and dAnhui Provincial Hospital,Anhui 230001, China

Edited by Philippa Marrack, Howard Hughes Medical Institute and National Jewish Health, Denver, CO, and approved November 28, 2012 (received for reviewApril 18, 2012)

Natural killer (NK) cells accumulate at the maternal–fetal interfacein large numbers, but their exact roles in successful pregnancyremain poorly defined. Here, we provide evidence that TH17 cellsand local inflammation can occur at the maternal–fetal interfaceduring natural allogenic pregnancies. We found that decidualNK cells promote immune tolerance and successful pregnancy bydampening inflammatory TH17 cells via IFN-γ secreted by theCD56brightCD27+ NK subset. This NK-cell–mediated regulatory re-sponse is lost in patients who experience recurrent spontaneousabortions, which results in a prominent TH17 response and exten-sive local inflammation. This local inflammatory response furtheraffects the regulatory function of NK cells, leading to the eventualloss of maternal-fetal tolerance. Thus, our data identify NK cells askey regulatory cells at the maternal–fetal interface by suppressingTH17-mediated local inflammation.

regulatory NK cells | fetomaternal tolerance

During pregnancy, allogeneic fetal cells invade the maternaldecidua but they are protected from the maternal immune

system. This invasion of extraembryonic trophoblasts does notharm gestation during normal pregnancy; it establishes toleranceat the maternal–fetal interface (1), (2), although the mechanismof such tolerance is not clear. In addition, inflammatory responsesinduced by a variety of mechanisms can result in embryo loss, butmild inflammation can be effectively controlled through regulatorymechanisms to maintain successful pregnancy (3). Thus, sup-pression of strong inflammatory responses is essential to ensurenormal pregnancy (4, 5), although the mechanisms involved inregulating local inflammation without compromising overall ma-ternal immunity during a successful pregnancy remain unknown.Multiple mechanisms are potentially involved in promoting im-

mune tolerance during pregnancy. For example, TH2 cytokine po-larization (6–9), the expression of the Fas ligand on trophoblastcells (10), and the inhibition of complement activation (11) arecrucial for ensuring tolerance at the maternal–fetal interface. Inaddition, a delicate balance exists between inhibitory (PD-L1, Stat3,and TGF-β1) and stimulatory (CD80 and CD86) signals during theestablishment of immune privilege (12–18). Furthermore, studieshave shown that galectin-1 (19) and indoleamine 2,3-dioxygenase(20) play pivotal roles in maternal–fetal tolerance. Several types ofimmune cells, such as CD4+CD25+ regulatory T cells, are alsoessential in the generation of maternal–fetal tolerance in mice andhumans (7, 21–24). Furthermore, natural killer (NK) T cells andimmature dendritic cells have been reported to promote the ex-pansion of Treg cells that confer protection of the fetus (19).Despite considerable progress, many questions remain unan-

swered. The most striking feature at the maternal–fetal interfaceis the accumulation of NK cells, which account for ∼60–90% ofimmune cells in the decidua in humans during early pregnancy(25–29) and are believed to be critical in maintaining immunebalance (4, 5, 25, 26, 30–32). Recent evidence has also shown

that decidual NK (dNK) cells play a key role in controlling tro-phoblast invasion and vascular remodeling (33, 34). AlthoughNK cells are an important cell type within the innate immunesystem and also act as sentinel cells in other models (35–37),their exact function at the maternal–fetal interface remains in-completely defined. Recent studies have reported that CD27,which belongs to the TNF receptor family, is an importantmarker in distinguishing different subsets of NK cells (38, 39).Based on the surface density of CD27 and CD11b, murine NKcells can be classified into four subsets that represent their dif-ferent maturation stages (40). We previously demonstrated thatthe expression of CD27 and CD11b can also identify distinctpopulations of human NK cells (39). Interestingly, we founda large number of CD27+ NK cells in the deciduas; these cellsare an important source of cytokines and show limited cyto-toxicity. However, the functions of this subset of NK cells inmaternal–fetal tolerance have not yet been identified.Here, we studied the function of dNK cells in successful preg-

nancy and examined NK subsets, their distribution, and cytokinesecretion profiles. We provide evidence that dNK cells possessa unique ability to maintain immune tolerance and suppress in-flammation by antagonizing TH17 cells that exist in natural allo-geneic pregnancies at the maternal–fetal interface. Moreover, thebalance between NK cells and TH17 cells is lost in patients withrecurrent spontaneous abortions. Our results suggest that NK cellsserve as pivotal sentinel cells that control local inflammation andmaintain tolerance at the maternal–fetal interface.

ResultsHuman CD56brightCD27+ NK Cells Preferentially Accumulate in theDecidua During the First Trimester. In contrast to NK cells in theperipheral blood, which accounted for a small fraction of the totallymphocytes (∼10%), NK cells were the dominant cell type in thedecidua during normal pregnancy (∼70%) (4, 25, 33, 41). In-terestingly, >90% of the NK cells in the decidua were CD56bright

NK cells, whereas less than 10% of the peripheral NK cells wereCD56bright NK cells (Fig. 1A).Because CD27 expression is known to define specific subsets of

NK cells (38–40), we compared the CD27 expression on CD56bright

dNK cells to that on peripheral NK cells, and we found that

Author contributions: B.F., X.L., Z.T., and H.W. designed research; B.F. performedresearch; R.S. contributed new reagents/analytic tools; X.T. and B.L. collected tissue sam-ples and information from patients; B.F., R.S., Z.T., and H.W. analyzed data; and B.F., X.L.,and H.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: ustcwhm@ustc.edu.cn or tzg@ustc.edu.cn.

See Author Summary on page 818 (volume 110, number 3).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1206322110/-/DCSupplemental.

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a much higher proportion of dNK cells were CD56brightCD27+ NKcells (29.84 ± 1.713%) (Fig. 1 B and C). We then investigated thekinetics of NK cells accumulating at the maternal–fetal interfaceduring pregnancy and confirmed that CD56brightCD27+ NK cellsaccumulated specifically during the first trimester. As the preg-nancy progressed, the percentage of this CD56brightCD27+ sub-set decreased significantly in the first trimester compared withthe subset in the term trimester (Fig. 1 D and E). Thus, theCD56brightCD27+ NK subset was well represented in the firsttrimester of pregnancy.

CD56brightCD27+ NK Cells Show Activated Phenotype and Are the MainSource of Cytokines. Due to the differential expression of acti-vating and inhibitory receptors, NK cells are phenotypically andfunctionally heterogeneous (42, 43). We compared the pheno-types of the CD27+ dNK and CD27− dNK cells and found theCD27+ dNK subset to express fewer inhibitory receptors, suchas CD158a, CD158b, KIR-nkat2, and KIR2DL3, and more ac-tivating receptors, such as NKp44, NKp46, and NKG2C. Thesefindings indicate that the human CD27+ dNK cell subset dis-plays a more activated phenotype than CD27− dNK cells (Fig. 2A and B). Comparison of CD27+ dNK cells and CD27+ pbNKcells showed limited differences in the expression of NKreceptors, suggesting that the CD27+ pbNK cells and CD27+

dNK cells were phenotypically similar, despite striking differ-ences their relative abundance (Fig. S1).Given that dNK cells are activated cells and lack significant

cytotoxic activity (30, 44, 45), we hypothesized that their main

function might be cytokine secretion. We sorted dNK cells usingFACS and stimulated them with phorbol 12-myrstate 13-acetate(PMA) and ionomycin in vitro; we found that IFN-γ was highlyproduced by dNK cells from multiple donors. Furthermore, theCD56brightCD27+ NK subset secreted much higher levels of IFN-γthan the CD27− subset. Approximately one-half of the CD27+

dNK cells (57.07 ± 5.301%) were IFN-γ–secreting cells (Fig. 2 Cand D). Thus, the main source of IFN-γ was the CD56brightCD27+

dNK cell subset.

NK Cells Control TH17 Cells and Maintain Tolerance During SuccessfulAllogeneic Pregnancy. T cells are also present in the decidua, wherethey can recognize alloantigens and become inflammatory. Pre-vious reports have shown that TH17 cells represent a barrier tothe induction of tolerance in transplantation and pregnancy (17,46, 47). To investigate the presence of TH17 cells, we firstassayed the IL-17–producing T cells present in the deciduaduring different phases of pregnancy. Of the CD3+CD4+ T cellsin the normal decidua during the first trimester, we found that2.107 ± 0.1521% of these cells secreted IL-17 (Fig. 3A). We alsofound that the mRNA expression levels of TH17-related factors(IL-17, RORγt, and IL-23R) were higher in first-trimester de-cidual lymphocytes than in full-term lymphocytes (Fig. 3 B–D).Thus, a small population of TH17 cells exists during the firsttrimester of normal human pregnancy.To determine whether these TH17 cells were induced following

contact with allogeneic antigens, we examined the potential roleof TH17 cells in spontaneous embryo loss using a mouse allogeneicpregnancy model. Allogeneic pregnant CBA/J × DBA/2 mice(CBA/J females mated with DBA/2 males) and syngeneic preg-nant CBA/J × CBA/J mice were monitored simultaneously. Ourdata showed that the percentage of TH17 cells among the totalCD4+ T decidual cells increased significantly in the allogeneicpregnant CBA/J ×DBA/2 mice at gestational day (gd) 14.5, whichsuggested that maternal T cells can respond to allogeneic fetalantigens and that decidual T cells have the potential to becomepathogenic TH17 cells. No differences were observed in the per-centages of TH17 cells in the spleens of either group (Fig. 3 E–G).Thus, in the allogeneic pregnancy model, a small population ofTH17 cells does exist at the maternal–fetal interface.To examine whether NK cells play a role in regulating TH17

cells in vivo, we deleted NK cells in pregnant CBA/J mice (allo-geneic mating) with anti-asialo GM-1 antibody (ASGM-1) (orPBS) at gd 0.5, 4.5, and 8.5. At gd 14.5, both groups were eu-thanized, and the deciduas and spleens from each group werecollected and analyzed separately. The efficiency and specificity ofNK depletion are shown in Fig. S2. The percentage of TH17 cells(IL-17A+CD4+CD3+T) in the deciduas was significantly in-creased in the NK-cell-depleted group (11.97 ± 0.9273%) com-pared with the control group (6.497 ± 0.5792%), suggesting a keyrole for NK cells in the suppression of TH17 cells during allogeneicpregnancy (Fig. 3 H and I). No significant differences were foundin the spleen cells between the groups (Fig. 3J). We repeated theexperiments in an allogeneic model in which Nfil3−/− femalesmated with BALB/c males, and Nfil3+/+ mice were used as con-trols. NK cells are absent in the Nfil3−/− mice. Both groups wereeuthanized at gd 14.5. The percentage of TH17 cells in the deciduaof Nfil3−/− mice was significantly higher than that in the Nfil3+/+

control group, further demonstrating that NK cells control TH17cells during allogeneic pregnancy (Fig. 3 K–M). Moreover, thenumber of live allogeneic fetuses per pregnancy decreased in boththe CBA/J allogeneic mouse model following NK cell depletionand in Nfil3−/− mice, suggesting that without NK cells, TH17 cellsmay have induced and caused fetal loss (Fig. 3 N–Q).

NK Cells Inhibit TH17 Cells Through an IFN-γ–Dependent Mechanism.Given the finding that CD56brightCD27+ dNK cells readily se-crete high levels of IFN-γ (Fig. 2 C and D) and that IFN-γ can

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Fig. 1. CD56brightCD27+ NK cells present in large numbers in humandeciduas of the first trimester. (A) Representative density plots showing ananalysis of CD56bright NK cells in gated CD56+CD3− NK cells isolated fromperipheral blood (pbNK) and decidua in the first trimester (dNK). (B) Rep-resentative density plots showing an analysis of CD27+ NK cells in gatedCD56bright pbNK and CD56bright dNK. (C) Percentages of CD56brightCD27+ NKcells in gated CD56bright pbNK and CD56bright dNK. n = 30 and 60 for pbNKand dNK, respectively. (D) Representative density plots showing an analysisof CD27 in gated CD56brightCD3− NK cells isolated from decidua in the firsttrimester and in the term trimester. (E) Percentages of CD56brightCD27+ NKcells in dNK in the first trimester and dNK in the term trimester. n = 62 and 6for dNK in the first trimester and dNK in the term trimester, respectively.The data in C and E are presented as the means ± SEM.

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inhibit the polarization of TH17 cells in mouse models (48, 49),we hypothesized that dNK cells may control TH17 cell polari-zation at least in part through the production of IFN-γ. First, weevaluated the effect of IFN-γ on human TH17 cell differentiationin vitro. We cultured freshly isolated CD4+ peripheral T lym-phocytes with or without the recombinant cytokines necessary forTH17 polarization, and we then quantified the production of IL-17and IFN-γ by ELISA. In medium supplemented with IL-1β andIL-23, a TH17 phenotype was induced, as indicated by the pro-duction of IL-17A. Similar to the results obtained in mice (48, 49),the expansion of human TH17 cells was inhibited by the additionof IFN-γ and was reinduced when IFN-γ was neutralized, whichsuggests that IFN-γ inhibits TH17 differentiation in humans.Second, we evaluated the effect of IFN-γ produced by activatedNK cells on human TH17 cell expansion. We obtained activatedNK cells by isolating peripheral NK cells from human donors andthen preactivated them for 12 h with IL-12 to induce IFN-γ ex-pression. These NK cells were then added to the TH17 polariza-tion system, and we found that TH17 cell differentiation wasinhibited when NK cells were added but was reinduced when IFN-γ was inhibited (Fig. 4A and Fig. S3). These data show that acti-vated human NK cells down-regulate TH17 differentiationthrough the production of IFN-γ.To verify whether the dNK-cell–derived IFN-γ acts directly on

TH17 cells, we obtained the deciduas from first-trimester normalpregnancies, sorted CD56+CD3− dNK cells, and cultured these

cells under different conditions for 60 h. Then, the supernatantfrom each culture was collected and added to the TH17 polari-zation system. After 6 d of culture, the supernatants were col-lected, and the concentrations of IL-17 were quantified byELISA. We found that the supernatants from normal dNK cellsinhibited TH17 expansion. In contrast, the supernatants fromnon-NK cells did not (Fig. 4 B and C). Furthermore, we alsopurified dNK cells and added them directly into TH17 inductioncultures. After 6 d of coculturing, the cells were briefly stimu-lated with ionomycin (1 μg/mL) and PMA (50 ng/mL) in thepresence of monensin (10 μg/mL) for 4 h, followed by in-tracellular cytokine staining for IL-17A. The supernatant fromthe culture system was also collected and analyzed by ELISA.Our data showed that dNK cells strongly inhibited developmentof TH17 cells. The addition of neutralizing anti–IFN-γ was ableto rescue the inhibition of TH17 induction (Fig. 4 D–F). Col-lectively, these results indicate that dNK cells directly antagonizeTH17 cells through an IFN-γ–dependent mechanism.

NK Cells Are Altered and Fail to Inhibit TH17 Cells in Patients withRecurrent Abortions. We obtained decidual tissues from patientswith recurrent spontaneous abortions and assessed whether therewere changes in the NK cells. We observed that the percentage ofNK cells decreased significantly in the deciduas of these patients(56.34 ± 4.379%) compared with the deciduas from healthypregnancies (66.06 ± 1.673%) (Fig. 5A). Given that the CD27+

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Fig. 2. CD56brightCD27+ NK are activated and are the main source of cytokines. (A and B) Percentage analyses of activating receptors and inhibitory receptorson gated CD27+CD56+CD3− dNK cells and CD27−CD56+CD3− dNK cells. n = 10 for KIR-nkat2, KIR2DL3, and NKG2C; 11 for NKp44, CD69, CD94, and NKG2A; 12for NKp46; 13 for CD158a and CD158b; and 15 for NKp30. Data in A and B are presented as means ± SEM. (C) Representative density plots of IFN-γ expressionon gated CD56+CD3−, CD27+CD56+CD3−, and CD27−CD56+CD3− dNK cells of the first trimester. (D) Percentages analyses of IFN-γ+ cells in dNK cells, CD27+ dNKcells, and CD27− dNK cells. n = 13 for each group.

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NK subsets were the main source of IFN-γ in normal pregnanciesand inhibited TH17 expansion (Figs. 2 and 3), we then investigatedthe ratio of CD27+ NK cells to TH17 cells and observed that thisratio was significantly decreased in this pathological state (Fig.5B). We further investigated additional cytokines, such as IL-10and IL-1RA, which are secreted by dNK cells. NK cells from

normal deciduas secrete large amounts of IL-10, which is the keycytokine responsible for suppressing TH17 cells (50). IL-1RA,which is a natural inhibitor of proinflammatory IL-1β, has alsobeen found in the supernatants of normal dNK cells. However, theexpression of these three cytokines (IL-1RA, IL-10, and IFN-γ)was significantly decreased in the dNK cells of patients who

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Fig. 3. NK cells control TH17 cells in normal allogeneic pregnancy. (A) Representative density plots showing analysis of IL-17–secreting cells in gatedCD3+CD4+ T cells in normal deciduas in the first trimester. (B) RNA levels of IL-17A expressions in decidual lymphocytes of the first trimester and the termtrimester. n = 8 and 4, respectively. (C) RNA levels of RORγt expressions in decidual lymphocytes of the first trimester and the term trimester. n = 8 and 4,respectively. (D) RNA levels of IL-23R expressions in decidual lymphocytes of the first trimester and the term trimester. n = 8 and 4, respectively. (E) Rep-resentative density plots showing analysis of IL-17–secreting cells in gated CD3+ T cells in deciduas and spleen from allogeneic pregnant CBA/J females andsyngeneic pregnant CBA/J females at gd 14.5. (F and G) Percentage analysis of IL-17+ cells in gated CD4+T cells from spleen and deciduas of allogeneicpregnant CBA/J females and syngeneic pregnant CBA/J females. n = 3 and 5 for allogeneic mating and syngeneic mating, respectively. (H) Representativedensity plots showing analysis of IL-17–secreting cells in gated CD4+ T cells in deciduas and spleen from allogeneic pregnant CBA/J females treated with PBS oranti–ASGM-1. NK cells were deleted by injecting 30 μL of ASGM-1 in 200 μL of PBS through the tail vein at gd 0.5, 4.5, and 8.5 each time. The control groupswere injected with 200 μL of PBS at the same time point as anti–ASGM-1 injection. Both groups were euthanized at gd 14.5 to examination the IL-17+ ex-pression and fetal resorption rate. (I and J) Percentage analysis of IL-17+ cells in gated CD4+T cells from spleen and deciduas of allogeneic pregnant CBA/Jfemales with treatment of PBS or anti–ASGM-1. n = 5 for both groups. (K) Representative density plots showing analysis of IL-17–secreting cells in gated CD4+

T cells in deciduas and spleens from allogeneic pregnant Nfil3−/− females or Nfil3+/+ females. Both group were mated with BALB/c mice and killed at gd 14.5. (LandM) Percentage analysis of IL-17+ cells in gated CD4+ T cells from spleens and deciduas of allogeneic pregnant Nfil3−/− females or Nfil3+/+ females. n = 4 forboth groups. (N and O) Representative picture of the number of embryos and live fetuses per uterus from allogeneic pregnant CBA/J females treated with PBSor anti–ASGM-1. (P andQ) Representative picture of the number of embryos and live fetus per uterus in allogeneic pregnant Nfil3−/− and Nfil3+/+ mice models.n = 4 for each group. The red arrows indicate embryos that were subject to hemorrhage, ischemia, and necrosis. The data in B–D, F, G, I, J, L, M, O, and Q arepresented as the means ± SEM.

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aborted (Fig. 5 C–E), suggesting that the regulation of NK cells inpatients with recurrent abortions is impaired. To identify thedistribution of dNK cells in this pathological state, we analyzedCD56+ cells by immunohistochemistry. The NK cells in normaldeciduas were consistently uniformly distributed, whereas thesecells were concentrated in the deciduas of patients who aborted(Fig. 5F). These results suggest that NK cells are abnormal in thedeciduas of patients who aborted; we observed not only differentpercentages of NK cells and levels of cytokine secretion but alsoabnormal localization of NK cells in these decidual tissues.We demonstrated that the supernatants from normal dNK cells

inhibited TH17 expansion through an IFN-γ–dependent mecha-nism. (Fig. 4 B–F and Fig. S3). To verify the direct regulatory roleof dNK cells in the pathological state, we next collected thesupernatants from dNK cell and decidual non-NK mononuclearcell cultures from patients with recurrent spontaneous abortionsunder different cytokine conditions and added them to the TH17cell polarization system. In contrast to normal NK cells, thesupernatants from the abnormal NK cells did not effectivelysuppress the expansion of TH17 cells (Fig. 6 A and B). These datasuggest that NK cells from patients who aborted are abnormal andno longer inhibit TH17 cells.

To further confirm the NK cell impairment, we obtained de-cidual tissues from patients with recurrent spontaneous abor-tions. In the deciduas of patients with recurrent spontaneousabortions, tissue inflammation was obvious, and the endometrialglands were disrupted and infiltrated by inflammatory cells (Fig.6C). We also found that the proportion of T cells was signifi-cantly increased in the deciduas of patients with recurrentspontaneous abortions (15.91 ± 2.761%) compared with those inpatients with normal pregnancies (6.500 ± 0.6407%) (Fig. 6 Dand E). We then determined whether TH17 cells were altered inthe deciduas of patients with abnormal pregnancies. FACSanalysis demonstrated that there were increased proportions ofIL-17–secreting CD4+ T cells in the deciduas of recurrentspontaneous abortion patients (10.64 ± 3.510%) compared withthose with normal pregnancies (1.707 ± 0.3891%) (Fig. 6F).Importantly, the TH17 cells were the main source of IL-17among the total T cells, and we found that CD4−CD3+ T cellssecreted very little IL-17 (Fig. 6D). Thus, during recurrentspontaneous abortions, increased numbers of TH17 cells andimpaired NK cells are present in the decidual tissues.

Expanded TH17 Cells in Decidual Tissues Cause Fetal Loss. To gainadditional insight into the underlying mechanisms, we isolated

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Fig. 4. NK cells inhibit TH17 cells through an IFN-γ–dependent mechanism. (A) ELISA of IL-17A and IFN-γ in cell-free supernatants of cultured T cells. The dataare representative of five experiments. CD4+T cells were cultured for 6 d in 96-well plates at a density of 1 × 105 cells per well in complete RPMI medium 1640and were activated with TH17 expansion condition. Purified NK cells from the same human donor were preactivated by IL-12 (100 U/mL). Culture supernatantswere collected after 6 d of culturing and analyzed by ELISA. (B) ELISA of IL-17A in cell-free supernatants of cultured T cells with supernatant from dNK cells.The data are representative of three experiments. CD56+CD3− dNK cells and non-NK mononuclear cells were separated and cultured in different conditionsfor 60 h. The supernatant from each culturing was collected and added into the system of TH17 expansion. After 6 d of culturing, each supernatant of theTH17 system was collected and the IL-17 concentration was quantified by ELISA. (C) ELISA of IL-17A in cell-free supernatants of cultured T cells with su-pernatant from non-NK decidual mononuclear cells. (D) Representative density plots of IL-17A expression in a TH17 expansion system in gated CD56−CD4+Tcells. Purified dNK cells from the first trimester were added to the TH17 expansion system at a ratio of dNK:T = 5:1. Cells were collected after 6 d of culturingand recultured under monensin (10 μg/mL), ionomycin (1 μg/mL), and PMA (50 ng/mL) for 4 h in complete RPMI medium 1640. Then, cells were collected forintracellular cytokine staining for IL-17A. (E and F) ELISA of IL-17A and IFN-γ in cell-free supernatants of the cultured dNK-T system. Supernatants werecollected after 6 d of culturing. The data are representative of six experiments. The data are presented as the means ± SEM.

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dNK cells and decidual non-NK mononuclear cells from patientswith recurrent abortions and examined the production of a panelof inflammatory cytokines. The secretion of IL-6 and IL-1β wasextremely low. However, the non-NK cells from the deciduas ofpatients who aborted produced high levels of IL-6 and IL-1β (Fig.7 A and B). Furthermore, using an antibody specific for IL-17,we found that the decidual tissues of patients with recurrentspontaneous abortions were infiltrated by much higher levels ofIL-17+ cells (Fig. 7C). The expression of IL-1 and IL-6, cytokinesthat support the development TH17 cells by non-NK cells in thedecidua, suggests extensive local inflammation, which may fur-ther affect the function of dNK cells.To determine whether there is a relationship between in-

creased frequencies of TH17 cells and fetal loss in vivo, weperformed the following experiments using a mouse model. First,naïve T cells were sorted from the spleens of C57BL/6 mice andadoptively transferred into pregnant mice at gd 7.5. In a differentgroup, the sorted naïve T cells were induced to develop intoTH17 cells for 6 d and then adoptively transferred into pregnantmice at gd 7.5. The two groups of pregnant mice were euthanizedat gd 14.5, and the fetal conditions were examined. We observedthat the group with adoptively transferred TH17 cells sufferedsignificant fetal loss; the rate of fetal loss was 83.60 ± 10.10%,which was significantly higher than that of the group that re-ceived nonpolarized naïve T cells (Fig. 7 D and E). Experimentsusing naïve T cells and TH17 cells from EGFP mice furtherconfirmed that TH17 cells could reach to deciduas and bedetected even 7 d after passive transferring (Fig. S4). Thus, TH17cells are highly pathogenic during pregnancy. Moreover, thenumber of live fetuses per pregnancy decreased in the CBA/Jallogeneic pregnant mice following NK cell depletion, which wasassociated with an increased fetus absorption rate, suggesting that

without the regulation of NK cells, TH17 cells expanded andcaused fetal loss (Figs. 7F and 3 N and O).

DiscussionOne striking feature of normal pregnancy is that a large pop-ulation of NK cells accumulates at the maternal–fetal interface(26–28). However, the exact immune function of these NK cellsremains incompletely defined. Our results demonstrate that dNKcells maintain immune tolerance by antagonizing TH17 cells.IFN-γ is closely involved in this effect, and the main source ofIFN-γ is confined to the CD56brightCD27+ dNK subset. Impor-tantly, the inhibition of TH17 cells by these NK cells is contingentupon the absence of local inflammation. We have provided ev-idence that this regulatory process is critical for successfulpregnancy and that NK cells play a key role in the control oflocal inflammation to ensure optimal fetal development at thematernal–fetal interface.NK cells are both phenotypically and functionally diverse (42),

and they have been shown to have many regulatory functions.TGF-β–secreting NK3 cells down-regulate T-cell activity andplay a key role in inhibiting the development of diabetes (51).The recently identified NK22 cells also have important regulatoryfunctions in mucosal immunity (52). Several elegant experimentshave revealed that human dNK cells can control trophoblast in-vasion and vascular remodeling by secreting an array of angio-genic factors, chemokines, and cytokines (33, 34, 53). NK cells inthe murine decidua have also been shown to be more cytotoxicduring episodes of severe inflammation (54). These studies, aswell as our previous report (39), have revealed the versatility,complexity, and tissue specificity of NK cells. However, the spe-cific immune function related to the accumulation of dNK cellsremains unclear.

A B

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*

Fig. 5. NK cells are abnormal in recurrent spontaneous abortion. (A) Percentages analysis of CD56+CD3− dNK cells from normal decidua and deciduas ofrecurrent spontaneous abortion. n = 67 and 11 for normal deciduas and deciduas of recurrent spontaneous abortion, respectively. (B) Percentages analysis ofthe ratio between CD27+CD56+CD3− dNK cells and TH17 cells from normal deciduas and deciduas of recurrent spontaneous abortion. n = 9 and 4, respectively.(C–E) ELISAs of IL-1RA, IL-10, and IFN-γ in cell-free supernatants of cultured dNK cells and decidual non-NK mononuclear cells. Decidual NK cells and non-NKcells were cultured in the presence of IL-15 (10 ng/mL) for 60 h in 96-well flat-bottomed plates at a density of 1 × 105 cells per well in complete RPMI medium1640. Data in A–E are presented as means ± SEM. (F) Immunohistochemistry for CD56+ cells in paraffin sections of normal deciduas and deciduas of recurrentspontaneous abortion. Dashed squares indicate matching areas that contain CD56+ cell clusters in each row that were magnified in the other frames andpresented in the corner insets. (Scale bar, 100 μm.)

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Here, we provide evidence that the CD56brightCD27+ NK subsetaccumulates at the human maternal–fetal interface and playsa key role in controlling local inflammation. In fact, in naturalallogenic pregnancies, mild inflammation and low levels of TH17cells can be developed at the maternal–fetal interface. Ourresults indicate that one of the functions of NK cells at thematernal–fetal interface is to inhibit local inflammation andmaintain immune balance. Additionally, the distribution of thissubset differed between the peripheral blood and the decidua,demonstrating that NK cells are tissue-specific and heteroge-neous. Moreover, we observed IFN-γ secretion by NK cells andfound that IFN-γ could down-regulate human TH17 cell expan-sion, as demonstrated in mice (48, 49). However, the effects ofIFN-γ are complex; results from mice have shown that IFN-γproduced by dNK cells also contributes to the remodeling ofdecidual arteries (55). The IFN-γ produced by dNK cells has alsobeen shown to be involved in the cross-talk between NK and

CD14+ cells in human decidua and the induction of Tregs (30).Furthermore, we revealed that high levels of IL-10, which alsohas a key role in suppressing TH17 cells, are secreted by normaldNK cells (50). The effects of these cytokines, including IFN-γ,IL-10, and IL-1RA, may be synergistic in regulating TH17 cellsand maintaining tolerance during pregnancy.The phenotype of NK cells can be altered during episodes of

severe inflammation. We selected recurrent spontaneous abortionsas a model of a pathogenic state. Severe stress, viral infection, andautoimmune disorders may also cause abnormal pregnancy andinflammation (3, 5). We found that the percentages of dNK cellsand the ratios of CD56brightCD27+ NK cells to TH17 cells de-creased, the regulatory cytokines IFN-γ and IL-10 were impaired,and the inhibition of inflammation was lost in this pathologicalstate. One possible explanation for this finding is that dNK cellshave increased cytotoxicity toward inflammatory cells (54) butimpaired regulatory capacity; another possibility is that after pro-

Donor 1 Donor 2

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Fig. 6. Abnormal NK cells fail to inhibit TH17 cells in recurrent spontaneous abortion. (A) ELISA of IL-17A in cell-free supernatants of cultured T cells withsupernatant from dNK cells of recurrent spontaneous abortion. Data are representative of three experiments. CD56+CD3− dNK cells and non-NK mononuclearcells were separated from patients of recurrent spontaneous abortion and cultured in different conditions for 60 h. The supernatant from each culturing wascollected and added into the system of TH17 expansion. After 6 d of culturing, each supernatant of the TH17 system was collected and quantified for IL-17concentration by ELISA. (B) ELISA of IL-17A in cell-free supernatants of cultured T cells with supernatant from non-NK mononuclear cells of recurrentspontaneous abortion. Data are representative of three experiments. (C) H&E staining for deciduas of normal deciduas and recurrent spontaneous abortion.(Scale bar, 50 μm.) (D) Representative density plots showing analysis of CD56 and CD3 expressions in lymphocytes, analysis of CD4 and CD3 expression in gatedCD56−CD3+ T cells, and analysis of IL-17 expression in gated CD4+CD3+ T cells and CD4−CD3+ T cells isolated from normal deciduas and deciduas of recurrentspontaneous abortion. (E) Percentages of CD56−CD3+ Tcells of lymphocytes isolated from normal deciduas and deciduas of recurrent spontaneous abortion.n = 72 and 10 for normal deciduas and deciduas of recurrent spontaneous abortion, respectively. (F ) Percentages of IL-17–secreting cells in the gated CD4+

T cells isolated from normal deciduas and deciduas of recurrent spontaneous abortion. n = 9 and 5 for normal deciduas and deciduas of recurrentspontaneous abortion, respectively. The data in A, B, E, and F are presented as means ± SEM.

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longed overactivation, NK cells may be immunodepleted andadopt an abnormal phenotype. It should be noted that duringsevere inflammation other cells in the decidua besides NK cellsmay also contribute to the loss of fetal tolerance via differentmechanisms. Treg cells are known to mediate maternal tolerancethrough IL-10 (56), which decreased significantly in specimensfrom spontaneous abortion (57). Furthermore, uterine DC frommice with high abortion rates displayed decreased expression ofIL-10 compared with that from mice with a low incidence offetal rejection (58). Alternatively, activated macrophages or M2macrophages also exert an immunosuppressive phenotype viathe production of IL-10 and indoleamine 2,3-dioxygenase ac-tivity (59). It would be interesting to determine how NK cellsinteract with such regulatory cell types during successful preg-nancy in future studies.We also present evidence for a direct relationship between

TH17 cells and fetal loss. TH17 cells participate in inflammatoryinfiltration in patients with recurrent spontaneous abortions (60);this finding is important because TH17 cells represent a criticallineage of proinflammatory T-helper cells involved in autoimmunedisease development (48, 49, 60, 61) and have been recognized asa barrier to the induction of tolerance in transplantation andpregnancy (17, 46). We also show that redundant TH17 cells di-rectly cause fetal loss in vivo, which suggests that pregnancy pa-thologies may be affected by treatments targeting TH17 cells. Wefurther show that increased levels of IL-6 and IL-1β induced aninflammatory microenvironment in the deciduas of patients withrecurrent spontaneous abortions that may promote the expansionand recruitment of TH17 cells. These observations provide im-portant clues for the pathogenesis of abnormal pregnancies andcould be useful in further clinical research.Our data suggest a model that during normal pregnancy dNK

cells act as sentinel cells to control local inflammation, which iscritical for maintaining tolerance at the fetal–maternal in-terface. However, this regulatory mechanism has limits, becauseNK cells can become altered by the inflammatory environment inthat the non-NK cells in the decidua secrete high levels of IL-1βand IL-6, which promote the expansion and recruitment of TH17cells. If inflammation is beyond the control of NK cells, toleranceis lost. These findings suggest a delicate role for dNK cells and theexistence of a critical balance between dNK cells and otherdecidual cells and immune cells, such as DCs (62) and Treg cells(7, 21, 23, 56, 57, 63), in both physiological responses andpathological responses.In summary, our data demonstrate that dNK cells can act as

sentinel cells to prevent local inflammation and maintain suc-cessful pregnancy at the human maternal–fetal interface. We haveshown that without NK cells, TH17 cells significantly contribute todecidual inflammation and abnormal pregnancies, and thereforeNK cells seem to maintain fetal–maternal tolerance by suppressingthe induction of TH17 cells. However, this role can be compro-mised in the presence of abnormal inflammation, which leads tothe loss of TH17 cell control and ultimately to the loss of tolerance.These findings may have important clinical implications.

Materials and MethodsHuman Samples and Cell Isolation. Decidua samples from normal pregnancies(n = 285) were obtained from elective pregnancy terminations. Twenty-fivedeciduas from abnormal pregnancies were obtained from patients withrecurrent spontaneous abortions (Table S1). Genetic or anatomical causes forabortions were excluded. The normal and abnormal samples were agedbetween 6 and 12 wk of gestation. Twenty term decidua samples werecollected after normal delivery. All of the decidua samples were collected

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Fig. 7. Prominent TH17 cells in decidua cause severe fetal loss. (A and B)ELISA of IL-6 and IL-1β in cell-free supernatants of cultured dNK cells anddecidual non-NK mononuclear cells. Decidual NK cells and non-NK cells werecultured in the presence of IL-15 (10 ng/mL) for 60 h in 96-well flat-bottomedplates at a density of 1 × 105 cells per well in complete RPMI medium 1640.(C) Immunohistochemistry for IL-17A in cryostat sections of normal deciduasand deciduas of recurrent spontaneous abortion followed by hematoxylincounterstaining. (Scale bar, 50 μm.) (D) Representative picture of embryosfrom naïve T-cell–transferred mice and TH17-cell–transferred mice. Naïve Tcells were sorted from C57BL/6 female mice and transferred directly via thetail vein at 1 × 106 per mouse or induced into TH17 cells under conditions forTH17 cell polarization. After 6 d of culturing, these cells were collected andtransferred via the tail vein at 1 × 106 per mouse. (E) Analysis of the re-sorption rate between the naïve T-transferred group and the TH17-trans-ferred group. n = 4 and 5, respectively. (F) Percentage analysis of the

resorption rate with or without NK depletion in allogeneic pregnant CBA/Jmodels. n = 7 and 12 for the PBS group and NK-depletion group, re-spectively. The data in A, B, E, and F are presented as the means ± SEM.

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from the Anhui Provincial Hospital. Peripheral blood mononuclear cells wereprepared from buffy coats obtained from healthy donors (Blood Center ofAnhui Province) by centrifugation through Ficoll. Before surgery, informedconsent was obtained from each patient. Ethical approvals were obtainedfrom the ethics committee of the University of Science and Technology ofChina. Isolation of decidua samples is discussed in SI Materials and Methods.

Murine Models of Pregnancy. Male and female C57BL/6 mice, male DBA/2,female EGFP C57BL/6 mice (8–10 wk old) were purchased from the ShanghaiExperimental Animal Center of the Chinese Academy of Science. Male andfemale CBA/J mice (8 wk old) were purchased from the Model Animal Re-search Center of Nanjing University. Nfil3−/− mice (without NK cells) werea generous gift from Tak Wah Mak (University of Toronto, Toronto, Can-ada). All animals were kept under specific pathogen-free conditions. All ofthe experimental procedures involving animals were conducted in accor-dance with the National Guidelines for Animal Use in Research (China), andpermission was obtained from the ethics committee at the University ofScience and Technology of China. Allogeneic CBA/J × DBA/2 mating combi-nations were established as follows: female CBA/J mice were mated in nat-ural cycling with male DBA/2 mice. The allogeneic Nfil3−/− × BALB/c matingcombinations were established as follows: female Nfil3−/− or Nfil3+/+ micewere mated in natural cycling with male BALB/c mice. Syngeneic CBA/J ×CBA/J mating combinations were established in an identical manner. For theTH17 cell transfer assay, syngeneic C57BL/6 × C57BL/6 mating combinationswere established. Detection of a vaginal plug was chosen to indicate day 0.5of gestation. For the NK depletion assay, allogeneic CBA/J × DBA/2 matingcombinations were established, and then the pregnant CBA/J mice wereinjected with 30 μL of anti–ASGM-1 (BD Biosciences) in 200 μL of PBS throughthe tail vein at gd 0.5, 4.5, and 8.5. Control animals were injected with 200 μLof PBS. Both groups were euthanized at 14.5 to examine the IL-17 expressionand fetal resorption rate.

Induction of Human TH17 Cells and the Effect of NK Cells. The CD4+ T cells wereisolated from the peripheral blood mononuclear cells of healthy donors bymagnetic bead depletion of CD19+, CD14+, CD56+, CD16+, CD36+, CD123+,CD8+, T-cell receptor-γ and T-cell receptor-δ-positive and glycophorinA-positive cells (CD4+T Cell Isolation Kit II; Miltenyi Biotec). The T cells werecultured for 6 d in 96-well round-bottom plates (Costar) at a density of 1 ×105 cells per well in complete RPMI medium 1640. The cells were activatedwith plate-bound anti-CD3 (5 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL;BD Biosciences) antibodies. Human IL-23 (25 ng/mL; EBioscience), IL-1β (25ng/mL; Peprotech), anti–IL-4(10 μg/mL; Peprotech), anti–IFN-γ (10 μg/mL;R&D Systems), and/or IFN-γ (100 ng/mL; Peprotech) were added at the startof the culture. The culture supernatants were collected after 6 d and ana-lyzed by ELISA. To determine the effect of NK cells on TH17 expansion, theNK cells were isolated from the same human donor as the CD4+ T cells andwere preactivated with IL-12 (100 U/mL), which was produced by our labora-tory, for 12 h before they were added to the culture system at different ratios.

Decidual NK Cell Culturing. Human dNK cells and non-NK mononuclear cellswere cultured in the presence of IL-15 (10 ng/mL; Peprotech) for 60 h in96-well flat-bottom plates at a density of 1 × 105 cells per well in complete

RPMI medium 1640. The supernatants from dNK cells and non-NK cells werecollected and used for the ELISA analysis to detect IL-1β, IL-6, IL-1RA, IL-10,and IFN-γ. For the assays to determine the effects of dNK cells on the ex-pansion of human TH17 cells, dNK cells and non-NK mononuclear cells werecultured under the following conditions: (i) IL-15 (10 ng/mL; Peprotech); (ii)IL-15 (10 ng/mL; Peprotech), IL-12 (100 U/mL, made by our laboratory), andIL-18 (50 ng/mL; Peprotech); and (iii) IL-15 (10 ng/mL; Peprotech) and low-dose PMA (10−7 M; Sigma). The cells were incubated for 60 h in 96-well flat-bottom plates at a density of 1 × 105 cells per well in complete RPMI medium1640. On the second day of TH17 cell culture, the supernatants from the dNKcells or non-NK mononuclear cells under different conditions were addedinto the TH17 cell expansion at a dose of 20 μL/well. Then, the supernatantsfrom the TH17 cell cultures were collected after 6 d of culturing and analyzedby ELISA for IL-17.

Passive Transfer of TH17 Cells in Vivo. Naïve CD4+T cells were isolated from thespleens of virgin female C57BL/6 mice by magnetic bead depletion of CD8a-,CD45R-, CD11b-, CD25-, CD49b-, TCRγ/δ-, and Ter-119-positive cells and thenby the positive selection of CD62L+ cells (CD4+ CD62L+ T Cell Isolation Kit II;Miltenyi Biotec). In the control group, 1 × 106 naïve CD4+ T cells wereresuspended in 200 μL of PBS and injected via the tail vein into pregnantC57BL/6 females on day 7.5 of gestation. In the TH17 cell transfer group,naïve CD4+ T cells were first incubated under conditions to induce polari-zation of the TH17 cells. The cells were activated with plate-bound anti-CD3(5 μg/mL; BD Biosciences) and anti-CD28 (2 μg/mL; BD Biosciences) anti-bodies. Mouse TGF-β (2 ng/mL), IL-6 (20 ng/mL), IL-1β (10 ng/mL), anti-mouse–IFN-γ (10 μg/mL), and anti-mouse–IL-4 (10 μg/mL), all purchased from R&DSystems, were added to stimulate the differentiation of the TH17 cells for 6d. The polarized cells were collected, and 1 × 106 cells were resuspended in200 μL of PBS and then injected via the tail vein into each pregnant C57BL/6female on day 7.5 of gestation. The recipient mice of both groups werekilled on day 14.5 of gestation, and the uteri were examined for the numberof healthy and resorbing embryos.

Quantitation of Embryo Resorption and Live Fetuses per Uterus. On day 14.5 ofgestation, the pregnantmice were killed and the uteri were examined for theresorption rate of the embryos. Resorbing embryos at this stage of gestationare subject to hemorrhage, ischemia, and necrosis and become smaller anddarker than the larger, viable, pink, healthy embryos. The resorbing rate wascalculated as follows: % resorbing rate = the number of resorbed embryos/the number of resorbed embryos and healthy embryos × 100. Live fetus peruterus was calculated as follows: Live fetus per uterus = the number of allfetuses per uterus − the number of resorbed embryos.

Statistical Analyses. We used paired two-tailed t tests (difference betweentwo groups) or unpaired two-tailed t tests to determine statistical signifi-cance (P < 0.05 was considered significantly different).

ACKNOWLEDGMENTS. This work was supported by Ministry of Science andTechnology of China 973 Basic Science Project Grants 2013CB530506,2009CB522403, and 2012CB519004 and Natural Science Foundation of ChinaGrants 81202367, 31021061, and 30730084.

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