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CHAPTER 8
NK Cells and PregnancyMikael Eriksson, Satarupa Basu and Charles L. Sentman
Abstract
Natural killer cells are found in large numbers in the endometrium and decidua, anddata suggest that NK cell functions and interactions with fetal-derived trophoblastscan have a profound impact on pregnancy. Altered NK cell numbers and activity have
been associated with a variety of clinical conditions such as endometriosis, recurrent pregnancyloss, and preeclampsia. Uterine NK cells have a unique phenotype compared to blood NK cellsand this is likely due to the specific tissue environment in which they reside. Specific chemokinesproduced by human endometrium and trophoblasts have been identified that may be respon-sible for recruitment of NK cells. Uterine NK cells can produce cytokines and may be animportant part of vascular remodelling during placental development. This chapter summa-rizes current knowledge of NK cells in the uterus and their role in pregnancy and reproductivedisorders.
IntroductionA mother’s immunological tolerance to a semi-allogeneic fetus has long been puzzling for
immunologists. The endometrium must be ready to respond to potential pathogen challenges,yet be able to control immune cell responses to allow the development of a semi-allogeneicfetus. Data have accumulated that there are extensive interactions between fetal cells and themother’s immune system. The immune system is often described as engaged in a struggle againstall kinds of invaders, including allogeneic fetal tissue. However, an alternative viewpoint gain-ing support is that the maternal immune system may work together with fetal derived cells tocreate a hospitable environment in which the fetus may develop.
Natural killer (NK) cells account for a substantial presence in the uterus and data suggestthat NK cell functions and interactions with fetal-derived trophoblasts can have a profoundimpact on pregnancy.1 This review summarizes current knowledge of NK cells in the uterusand their role in pregnancy and reproductive disorders. We have focused on our understandingof human uterine NK (uNK) cells and also discuss particularly noteworthy findings from ani-mal studies. Valuable data have been derived from animal studies, but significant differencesexist in the structure of the placenta between species. Human NK cells are present in nonpreg-nant endometrium and are found throughout the endometrium and decidua, while NK cellsare absent in mice prior to implantation and are restricted to the mesometrial triangle anddecidua basalis. In humans, decidualization occurs during every menstrual cycle and there isextensive invasion of trophoblasts into maternal tissues. Decidualization occurs in mice afterimplantation, and trophoblast invasion into maternal tissue is minimal. These differences makeextrapolation of data between species very difficult, so conclusions made in one species will notalways be applicable to another.2
Immunology of Pregnancy, edited by Gil Mor. ©2005 Eurekah.comand Springer Science+Business Media.
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Immunology of Pregnancy2
NK cells, also referred to as large granular lymphocytes or LGLs, have the ability to sponta-neously kill tumor cells and secrete cytokines that can have powerful effects on immune- andnonimmune cells. As part of the innate immune system, NK cells are believed to act as sentinelcells prepared to attack foreign pathogens and promote host immune defenses.3-5 Altered NKcell numbers and activity have been associated with a variety of clinical conditions involvingreproductive organs and reproductive failure. Reduced NK cell activity is associated with anincrease in the incidence of ovarian cancer, uterine cancer, and endometriosis.6-9 Higher NKcell activity has been associated with recurrent pregnancy loss,10,11 while NK cell function andspecific NK cell receptors may be involved in the development of preeclampsia.12,13 Thus,proper NK cell function in the uterus is an integral part of successful pregnancy.
Uterine NK CellsHuman NK cells are found in the blood, lymphoid organs, liver, and various mucosal tis-
sues including lung, intestine, and uterus. Human peripheral blood NK cells can be dividedinto two major subsets based on the density of CD56 expression. CD56dim cells account for90% of blood NK cells and have a high spontaneous lytic activity against tumor cells. This NKcell subset expresses CD16 (FcRγIII), killer cell immunoglobulin-like receptors (KIRs) andmany of these cells express CD57.14 The CD56bright NK cells account for approximately 10%of blood NK cells and these cells have less spontaneous lytic activity and have a high capacity toproduce proinflammatory cytokines upon stimulation with monokines.15 This NK cell subsetexpresses CD94/NKG2A/C dimers but contains only a small percentage of cells that expressCD16 or KIRs and these cells lack CD57 expression.16 Although comprising only a fraction ofNK cells found in the peripheral blood, the CD56bright subset is the primary NK cell subsetfound in lymph nodes.17,18 In addition to the markers mentioned above, there are a largenumber of other cell surface molecules that can be used to further subdivide NK cell sub-sets.16,19
Uterine NK cells have a unique phenotype compared to blood NK cells. uNK cells havepreviously been described as endometrial granulocytes, granular endometrial stromal cells, Kcells, or even decidual NK (dNK) cells.20 NK cells in the uterus express CD56 and CD94, fewexpress CD16, and none express CD57 or CD8.21,22 Most CD57+ cells in the endometriumare T cells.23 A significant percentage of uNK cells express KIRs on their cell surface.22,24
Unlike blood NK cells, uNK cells express CD9 and CD69 on their cell surface.22,25 Thus uNKcells have a cell surface phenotype that is unique. A summary of molecules and receptors onuNK- and blood NK cells is shown in Table 1. Recent molecular analysis of NK cells fromdecidua using gene array technology revealed 278 genes that were differentially expressed be-tween decidual NK cells and blood NK cells.25 This study indicated that decidual NK cellswere more similar in their gene expression profile to CD56bright NK cells than to CD56dim NKcells. It remains unclear whether decidual NK cells are derived from blood NK cells that havedifferentiated in the unique environment of the decidua, or if they represent a special lineage ofNK cells that are selectively produced for the decidua. Most evidence seem to favor the ideathat the NK cells in the decidua are derived from uNK cells in the endometrium at the time ofimplantation or from newly recruited blood NK cells. These NK cells may respond to externalsignals to change their gene expression profile and presumably their function to respond to theunique requirements of the decidua. NK cells do not express receptors for estradiol and proges-terone, so the action of sex hormones on NK cell function or recruitment is likely mediated viahormone action on other cells, such as fibroblasts or epithelial cells.26,27 One report indicatedthat human uNK cells express glucocorticoid- and estrogen-β1 receptors but it is not known ifthis estrogen receptor functions in uNK cells.26 NK cells isolated from the nonpregnant en-dometrium and decidua may not represent distinct phenotypes, but there may be a continuousprocess of differentiation such that the phenotype and functional activity of uNK cells maychange as the local environment adapts to alterations in hormone levels and changes in thestromal cell environment.
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3NK Cells and Pregnancy
One complication is that some studies have used NK cells isolated from first term deciduawhile others study NK cells from nonpregnant endometrium, and the hormonal status andlocal tissue microenvironment can be quite different between endometrium and decidua. En-dometrial uNK cells are isolated from hysterectomy tissue or from microcurettage scraping ofthe endometrial lining. Even for endometrium from nonpregnant women, NK cells found inthe proliferative phase and those from the secretory phase are in distinct tissue microenviron-ments and hormonal conditions. Thus, when reading the literature care must be taken tounderstand the tissue environment from where uNK cells have been isolated.
Recruitment of NK Cells into the Endometrium and DeciduaThe human uterine endometrium is a complex mucosal tissue that is regulated by sex hor-
mones throughout the menstrual cycle.34,35 NK cell numbers are low early in the proliferativephase and increase as the menstrual cycle progresses.36-38 NK cells may account for up to 70%of the leukocytes in the endometrium late in the secretory phase. At least two possible mecha-nisms have been hypothesized to be responsible for the drastic increase of uNK cells within theuterus: either in situ proliferation and/or a selective recruitment from the peripheral NK cellpool have been suggested.34,39,40 Although proliferation could explain some of the increasednumbers of NK cells, an active recruitment of these cells to the uterus is likely to play a majorrole. Data from murine studies indicate that uNK cells are derived from blood cells or bonemarrow cells and not from NK cells within the uterus,41 although in humans it has not beenruled out that NK cells may be renewed from a precursor within the noncycling part of theendometrium.
Table 1. Comparison between uterine NK (uNK) cells, CD56bright and CD56dim bloodNK cells
Markers uNK cells a Blood CD56bright Blood CD56dim
CD56 ++b ++ +CD16 +/- +/- +CD69,CD9,CD151 + - -L-selectin - ++ +/-CD57 - - +CD49a ++ - -CD44 ++ ++ +PEN5-PSGL1 +/- - +CD94 ++ ++ +/-KIRc + +/- +KIR/NKAT 2 ++ - ++NKG2E/C/A ++ + +/-CXCR1 - - ++CXCR2 - - +CXCR3 ++ ++ +CXCR4 + ++ ++CXCR5 - - -CCR1,CCR2,CCR3,CCR4 - - -CCR5,CCR7 + ++ -CX3CR1 +/- ++
a Some studies used NK cells from endometrium and others used decidual NK cells; b++: highexpression; +: low expression; +/-: expression on subsets; -: no expression; cKIR = Killer cellimmunoglobulin-like receptor family. References 15, 25 and 28-33.
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Immunology of Pregnancy4
Given the increase in NK cells during the menstrual cycle and the role of sex hormones inmodifying changes in endometrium, it has been proposed that sex hormones may regulate NKcell recruitment and expansion in the endometrium. Migration of cells into tissue involves avariety of molecules, including adhesion molecules and chemokines. Cells in blood must bindto the endothelium and firmly adhere so that they can follow chemotactic gradients into tis-sues. Many blood cells express several different chemokine receptors and more than one maybe involved in proper migration of cells into tissues. NK cells are found widely within nonpreg-nant endometrium and they can be associated with other leukocytes in small aggregates.36,42,43
In the first trimester of pregnancy, uNK cells accumulate as a dense infiltrate around the tro-phoblast cells and spiral arteries.20 As pregnancy progresses, uNK cells disappear from thedecidua and are absent at term. These findings suggest that specific signals are involved in therecruitment and localization of NK cells within the uterus.
Blood NK cells express a variety of chemokine receptors, such as CCR5, CXCR3, andCXCR4, and specific migration of NK cells has been induced by chemokines in vitro.31,38,44
uNK cells have been shown to express CXCR3, CCR5, and CCR7, but not other chemokinereceptors.29 Not all chemokine receptors have been analyzed on uNK cells and low levels ofreceptor expression may be functionally relevant. Kitaya and colleagues have examined theexpression of chemokines within the endometrium at different stages of the menstrual cycle.They have reported that the expression of CCL4, CXCL9, and CXCL10 increases during themenstrual cycle and correlates with the increase in the number of NK cells in the en-dometrium.45,46 The authors analyzed the expression of these chemokines by immunohistochem-istry and found chemokines expressed in epithelial and perivascular stromal cells. It has beendemonstrated that estrogen and progesterone are able to induce the expression of CXCL10 andCXCL11, but not other chemokines in primary human endometrium.29 Several potentialchemokine receptor-ligand pairs have been described in the decidua that could be involved inthe leukocyte trafficking. Trophoblasts have been reported to express CXCL12, a ligand forCXCR4.30 This study also shows that in vitro, CXCL12 preferentially recruits CD56bright NKcells compared to CD56dim NK cells. Placenta and endometrium have been shown to produceCCL3 that can recruit blood NK cells.47,48 It has also been reported that CD16-/CD56bright
NK cells express chemokine receptor CCR5 and that its ligand CCL4 acts as a strongchemoattractant for these cells.49 However, murine studies have shown normal uNK cell re-cruitment in mice deficient in CCR2 or CCR5.50 Thus, a large number of studies show that theendometrium and decidua can produce chemokines that can recruit NK cells. Which chemokinesare involved in NK cell recruitment in vivo remains to be determined, and it is possible thatmore than one chemokine is involved.51 For example, CXCL10 induced by sex hormones inendometrium prior to implantation may recruit NK cells into the endometrium. As tropho-blasts invade the decidua, chemokines derived from trophoblasts (e.g., CXCL12) may recruituNK cells to reorganize placental arteries and facilitate trophoblast invasion of maternal tissue.
Studies have identified specific adhesion molecules involved in NK cell attachment to uter-ine endometrium. It has been established that L-selectins and α4 integrins are important forthe binding of lymphocytes possibly facilitating extravasation into tissues.52-55 Adhesion ofCD56bright cells to uteri from pregnant or hormone-treated ovariectomized mice was enhancedthrough L-selectin- and α4 integrin-dependent mechanisms.56 The ability of human CD56bright
NK cells to adhere to mouse uteri is associated with successful outcome of in vitro fertilization(IVF) treatment.57 The binding of NK cells also involved VCAM-1 and peripheral lymph nodeaddressin. VCAM-1 is expressed at the site of trophoblast invasion and might allow NK cells tomigrate continuously to these sites.56,58,59 The proper migration of NK cells into the endometriumand localization to specific areas may be critical for NK cell function in the uterus.
Function and Regulation of uNK CellsHuman decidua contains a large number of NK cells and many of them localize near
trophoblasts. Uterine NK cells have been proposed to have several different functions in
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5NK Cells and Pregnancy
pregnancy, including to: (i) help shield trophoblasts bearing paternal antigens from the ma-ternal immune system, (ii) protect the mother from trophoblast invasion and limit expansionof trophoblasts, (iii) be involved in regulation and restructuring of maternal spiral arteries,and (iv) be part of the innate defense system and protect against infection in the uterus. Theseideas are not mutually exclusive and there is evidence to support each of them.
Regulation of uNK CellsIt has been proposed several years ago that successful pregnancy was associated with Th2-type
environment rather than a Th1-type environment.60 It has become clear that the reality is morecomplex, and there is evidence that both Th1 and Th2 cytokines are produced in decidua. Likeblood NK cells, uNK cells can produce IFN-γ, GM-CSF, IL-10, TGF-β1, and IL-8 amongother cytokines.22,61-63 One study indicated that fresh decidual NK cells produce cytokinesspontaneously.64 uNK cell derived cytokines may have significant effects on decidualizationand trophoblast invasion. Human endometrium expresses a wide range of angiogenic growthfactors, and uNK cells may play an important role in abnormal endometrial angiogenesis.65
uNK cells produce cytokines that are not normally made by blood NK cells such as angiogenicgrowth factors and leukemia inhibitory factor (LIF).61,65 LIF is considered an essential cytokinefor implantation in mice.66
Many different cytokines that alter NK cell function have been shown to be present inthe human endometrium.67,68 Endometrium is a source of both IL-15 and prolactin,69-71
and both of these have been implicated in the proliferation and differentiation of uNK cells.There are data that uNK cells express prolactin receptors.72,73 Evidence suggest that IL-15 isrequired by uNK cells for survival and proliferation.74-78 In humans, IL-15 is present through-out the menstrual cycle, is increased during the secretory stage and early pregnancy, and isproduced by stromal cells during decidualization.69,79-81 It has been suggested that IL-15expression in endothelium may be important for NK cell attachment, and perhaps IL-15expression by decidual endothelium may be involved in specific localization of uNK cellsclose to spiral arteries.59,82,83
Several reports have observed effects of TGF-β on NK cells.84-88 Members of the TGF-βfamily are powerful immunoregulatory molecules that act on a range of different immunecells and can demonstrate both activating and inhibitory function.89-92 TGF-β proteins areproduced as inactive precursors that bind to extracellular matrix and cell surface proteinswhere they can be activated.93 The mechanisms that regulate and activate TGF-β proteinsremain unclear. A recent paper shows that endogenous TGF-β-mediated inhibition is a mecha-nism that regulates uNK cell-derived cytokine production.22 Another study reported thatCD56+ lymphoid cells in human first trimester pregnancy decidua are a source of noveltransforming growth factor-β2-related immunosuppressive factors.94 Furthermore, data sug-gest that TGF-β1 may be necessary to maintain pregnancy but may also be a risk factor forrecurrent miscarriages.95
Molecules that differentiate NK cells in the endometrium are poorly understood. IL-11 hasbeen implicated in the differentiation of uNK cells. IL-11 deficient mice have been shown tolack NK cells at implantation sites.96 Although NK cell precursors could home to the uterus ofIL-11 receptor α-/- mice, the NK cell defect was not thought to be due to the NK cells them-selves but to signals within the environment. This study showed that the requirement for IL-11to induce NK cell differentiation was tissue specific and that IL-11 signaling indirectly affecteduNK cell differentiation. Many other factors are likely involved in uNK cell differentiation andthey remain to be discovered.
NK Cell Trophoblast InteractionsNK cells and macrophages are found near trophoblasts in the first trimester of pregnancy.20,97
Due to trophoblast expression of paternal antigens, low MHC expression, and their associationwith NK cells, it has been theorized that the trophoblast-NK cell interaction is a key part in the
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Immunology of Pregnancy6
control of placental development. Trophoblasts express three different HLA molecules thatcan interact with NK cell receptors, HLA-C, HLA-E, and HLA-G. CD158 receptors (mem-bers of the KIR family) bind to different HLA-C alleles and data suggest that thisreceptor-ligand interaction is involved in development of preeclampsia (see below). CD94/NKG2A dimers recognize HLA-E, and KIR2DL4 is expressed on uNK cells and recognizesHLA-G molecules. Many of these receptor-ligand interactions have been shown to alter NKcell function.
Much effort has focused on the susceptibility of trophoblasts to NK cell mediated cytotox-icity. Most studies have indicated that human trophoblasts are resistant to NK cell mediatedkilling.98,99 Since most decidual NK cells express CD94/NKG2A dimers, it has been suggestedthat uNK cells could recognize HLA-E expressed on trophoblasts and inhibit NK cell activa-tion. However, lysis of trophoblasts could not be induced even after blocking CD94/NKG2receptors on NK cells or MHC class I molecules on trophoblasts.98 Removing MHC class I ontrophoblasts by acid treatment could not induce lysis by decidual NK cells. Lysis of tropho-blasts could only be observed using IL-2 activated decidual NK cells. One study with murinetrophoblasts demonstrated trophoblast rejection in nonpregnant allogeneic mice at extra-uterinesites, supporting the idea that trophoblasts are not inherently resistant to NK cells but thatlocal factors in decidua protect them from immune attack.100 The inability of trophoblast cellsto act as target cells for NK cell cytotoxicity, even when known inhibitory receptors were blocked,implies that alternative inhibitory pathways exist or that trophoblasts are unable to triggeruNK cell activating receptors.
HLA-G is only expressed by trophoblasts and can interact with KIR2DL4 receptors onuNK cells. Data have suggested that HLA-G may inhibit NK cell activation, but ligation ofKIR2DL4 led to stimulation of IFN-γ but not the triggering of cytotoxicity.101 A recent pub-lication suggested that membrane-bound HLA-G is able to stimulate purified uNK cells whileit suppressed unfractionated mononuclear cell effector functions.102 Because IFN-γ produc-tion by uNK cells is believed to be important for vascular remodelling (see below), the recogni-tion of HLA-G on trophoblasts by uNK cells may be important for placental development.The fact that trophoblast protection from NK cell lysis appears to be largely independent ofHLA class I expression suggests a modulatory function for HLA-E and HLA-G rather than astrict inhibitory effect on uNK cell effector functions. These receptor-ligand interactions maymodulate uNK cell production of cytokines and angiogenic factors promoting trophoblastinvasion and differentiation or tissue remodelling.65
NK Cell Regulation of Spiral ArteriesKey observations in murine studies have been made using mice deficient in NK cells or
specific effector molecules. NK cell deficient mice do have offspring, but the litters are smallerin number. Although it remains unclear how much can be extrapolated to human placentaformation, careful analysis demonstrated ultra-structural consequences for implantation andplacental formation in the absence of uNK cells in NK cell deficient mice.41,103 Failure tosustain decidual integrity and loss of spiral artery modifications were the two key featuresaffected in these mice. Additional studies also suggested that uNK cell derived IFN-γ wasnecessary for the proper structure of spiral arteries and placental formation.104 In humans,uterine NK cells are nearly absent by 20 weeks of gestation, a time when vascular changes arecomplete. In the placenta, uNK cells are often seen in close proximity to transformed andnontransformed spiral arteries. Moreover, uNK cells can be a source of angiogenic growthfactors such as NO synthase or Ang2 that may be involved in destabilizing blood vessel struc-ture and promoting blood vessel remodelling.65,105 The role of uNK cells in remodelling hu-man spiral arteries remains unknown, but data suggest involvement of NK cell receptors in thedevelopment of preeclampsia that supports a role for human uNK cells in vessel remodelling(see next section).
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7NK Cells and Pregnancy
NK Cells in Reproductive Disorders
Recurrent Pregnancy LossRecurrent pregnancy loss (RPL) is defined as three or more consecutive spontaneous abor-
tions with the same partner. About half of all cases are explained by universally accepted aeti-ologies. Thus, proposed immune based aetiologies are being studied as a possible explanationfor some of the other cases. Activated CD56+ NK cells are present in increased numbers inperipheral blood in women with RPL.106-110 NK cells produce IFN-γ which is a Th1 cytokinethat is associated with RPL,111,112 and LIF is down-regulated by IFN-γ.113 It has been demon-strated that CD56bright NK cells are decreased in the secretory phase endometrium in patients withunexplained recurrent miscarriage114 and in vitro fertilization-embryo transfer failure.115 One possibilityis that some of these patients have an influx of CD56dim NK cells that respond against fetalcells. There is evidence that NK cells and PBMCs from women with unexplained RPL respondto trophoblast extracts in vitro by proliferating and releasing embryotoxic factors that adverselyaffect embryo growth, while cells from women with normal pregnancies do not show such aresponse.116 An increase in the absolute count of activated NK cells (CD56dim/CD16+/CD69+)in the peripheral blood is associated with a reduced rate of embryo implantation during IVFtreatment. Furthermore, women with high numbers of CD56dim/CD69+ blood NK cells whowere able to achieve pregnancy had a significantly higher rate of miscarriage.10 However, onemust remember that the local maternal immune response to the fetus-derived cells may not besimilar to the systemic response against fetal cells. Correlative data implicate NK cells in RPL,but a direct link has not been established. It is unclear whether an increase in NK cell cytotoxicactivity and/or a decrease in other NK cell functions may account for some cases of RPL.
EndometriosisEndometriosis is a condition where there is an ectopic occurrence of endometrial tissue,
often in the peritoneal cavity. One theory is that endometrial cells move into the peritonealcavity due to aberrant menstrual flow, and women with endometriosis frequently have diffi-culty becoming pregnant. Decreased NK cell activity in peripheral blood and peritoneal fluidof women with endometriosis has been established.117 One mechanism for decreased NK cellactivity may be increased KIR expression on NK cells, because increased expression of KIRs isgenerally associated with decreased NK cell activity.8,9,118 Thus, NK cells may help prevent thedevelopment of endometriosis by removing endometrial cells in the peritoneum.
PreeclampsiaPreeclampsia is a serious complication of pregnancy and leads to high maternal blood pres-
sure, elevated concentrations of urinary protein and poor fetal growth.119,120 It can be causedby inadequate remodelling of spiral arteries and lead to the life-threatening condition of ec-lampsia. Alterations of NK cell function may be important in failure to reorganize blood ves-sels at the maternal-fetal interface and account in part for preeclampsia.13 There is an increasedincidence of preeclampsia in mothers who lacked most or all activating KIR genes (the AA KIRgenotype) when the fetus possessed HLA-C genes of the C2 subgroup.12 This combinationleads to the greatest inhibition of NK cells via KIR-HLA-C interactions. These data supportthe idea that without the presence of activating receptors on NK cells, these cells may be pre-vented from completing the task of vasculature remodelling and result in preeclampsia.
Other studies suggest that preeclampsia is associated with a Th1/Th2 imbalance with apredominant Th1 immunity.121 This may be considered a failure of tolerance (i.e., a decreasein NK cell inhibition) or an increase in NK cell activity. Serum levels of granulysin, a cytotoxicgranule protein of natural killer (NK) cells and cytotoxic T lymphocytes, were significantlyelevated in preeclampsia patients when compared to those in normal pregnancy subjects.13
Preeclampsia was associated with an increased number of CD56dim NK cells in umbilical cordblood compared to the control group.122 Thus, aberrant NK cell activity may have a significantrole in the pathogenesis of this disorder.
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Immunology of Pregnancy8
SummaryNatural killer cells are found in large numbers in the endometrium and decidua and data
suggest that NK cell functions and interactions with fetal derived trophoblasts can have aprofound impact on pregnancy. Uterine NK cells have a unique phenotype compared to bloodNK cells and this is likely due to the specific tissue environment in which they reside. We arebeginning to understand the role of uNK cells, their cytokines and key receptors that allowthem to function to establish a functional and supportive maternal-fetal interface. Data exist toimplicate both excessive NK cell activation and the lack of NK cell function in reproductivedisorders. This suggests there is a delicate balance that must be maintained where fetal-derivedtrophoblasts and maternal decidual cells work together to allow proper placental developmentand fetal growth.
AcknowledgementsThis work was supported in part by a grant from the National Institutes of Health (AI51877).
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