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Review
. 2021 Jun 21:12:607669.
doi: 10.3389/fimmu.2021.607669. eCollection 2021.

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Oisín Huhn  1   2 , Xiaohui Zhao  2 , Laura Esposito  2   3 , Ashley Moffett  2   3 , Francesco Colucci  1   2 , Andrew M Sharkey  2   3
Affiliations
Review

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Oisín Huhn et al. Front Immunol. .

Abstract

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

Keywords: decidua; endometrium; innate lymphoid cell; placenta; pregnancy; tissue resident natural killer cell; uterine natural killer cell.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Possible roles of uterine NK cells and ILCs in human endometrium and placental development in humans and mice. (A) Important biological processes in endometrium during the human menstrual cycle are shown, which may be influenced by local immune cells. Following menstruation, the endometrium undergoes repair and proliferation under the influence of oestrogen from the ovary and blood supply is re-established involving both spiral arterioles and new capillaries. Following ovulation, progesterone action results in decidualization of the endometrial stroma and a rapid increase in the number of uNK cells, which comprise up to 70% of the leukocytes (shown in green) towards the end of the cycle. Dynamics of other ILC populations in human endometrium through the cycle are less well understood. Murine endometrium cycles by exposure to oestrogen then progesterone but the estrous cycle only lasts ~5 days and there is no menstruation. (B) The human maternal-fetal interface. Spiral arterioles (SpA) branch from the radial arteries and penetrate the decidua to supply the placenta. Right hand panel shows placental villi bathed in maternal blood which enters the intervillous space. Placental villi are covered by syncytiotrophoblast, beneath which is the villous cytotrophoblast layer (VT). Anchoring villi attach to decidua via columns of extravillous trophoblast (EVT) which differentiates from VT. Some EVT migrates down spiral arteries as endovascular trophoblast (ENDT) while other EVT cells migrate into the decidua as far as the myometrium where they transform into trophoblast giant cells (TGC). Interstitial EVT home to maternal spiral arteries and participate in their remodelling to provide a low pressure, high capacity blood supply to the placenta. During the first half of pregnancy, when spiral artery remodelling occurs, trophoblast invasion in humans is much deeper and more extensive than in mice. EVT invading into the decidua encounter maternal leukocytes (shown in green). In humans in the first trimester, these comprise: ~70% NK and ILCs, 20% macrophages and ~10% T-cells, which include CD4 and CD8 cells. Figure (B) adapted from Moffett and Colucci (1). (C) Schematic view of pregnant mouse uterus showing multiple implantation sites. Radial arteries branch from the arcuate artery to supply each developing fetus. The right hand panel shows a uterine cross section cut as indicated by the dotted line, with the arrangement of the placenta and decidua at gestation day (gd) ~12.5. The radial arteries penetrate through the myometrium, traversing a specialized structure that develops around mid-gestation, known as the mesometrial lymphoid aggregate of pregnancy (MLAP). The MLAP is not present in humans. Spiral arteries (SpA) branch from the radial artery into the decidua, rich in uterine NK cells but then merge at the interface between the placenta and the decidua to form a large blood canal that supplies the labyrinthine layer of the placenta where gaseous exchange takes place between maternal and fetal blood. This blood canal is lined with specialized fetal trophoblast cells (shown coloured purple). The boundary between the placenta and decidua is delineated by trophoblast giant cells (TGC), which show minimal invasion into the decidua - those few that do invade are largely perivascular. From gd12.5, glycogen rich trophoblast cells invade more extensively into the decidua (later than timepoint shown here), but spiral artery remodelling is largely complete by gd12.5. Panel C based on data from Adamson et al. (2).
Figure 2
Figure 2
Development and functions of NK cells and ILCs Simplified overview of key stages of ILC development. Scheme shown is largely based on murine data. Table shows typical cytokines that stimulate the main ILC subsets, together with their principal effector molecules and known immune functions. Phenotypic markers commonly used to identify human subsets are shown and assumes prior gating on lin-CD45+ cells. CLP common lymphoid progenitor, CILP, common innate lymphoid progenitor; CHILP, common helper ILP; ILCP, innate lymphoid cell precursor; NKP, NK progenitor; LTiP, lymphoid tissue inducer precursor; LTi, lymphoid tissue inducer; NFIL3, Nuclear factor, interleukin-3 regulated; ID2, inhibitor of DNA binding 2; GATA3, GATA binding protein 3; PLZF, promyelocytic leukemia zinc finger; T-bet, T-box transcription factor 21; Eomes, eomesodermin; AhR, aryl hydrocarbon receptor; ROR, Retinoic acid–related orphan receptor; TSLP, thymic stroma lymphopoietin; Areg, amphiregulin; LT, lymphotoxin; IFNg, interferon-gamma; IL, interleukin; NCR, natural cytotoxicity receptor; NK, natural killer. Figure and phenotypic markers adapted from Vivier et al. (18) and Guia and Mancinelli (19).
Figure 3
Figure 3
Major peripheral blood (pbNK) and uterine NK cell (uNK) subsets in human and mouse, with possible trophoblast MHC ligands for selected uNK receptors. The two main human pbNK subsets are Lin-CD56bright (~10% of total NK) and CD56dim (~90%); in mice the equivalent NK subsets are lin-CD27+CD11b+ and CD27-CD11b+ (56). In the human uterus, uNK cells are defined as lin-CD56+CD49a+ (or CD56+CD9+, since both CD49a and CD9 are markers of tissue residency in the uterus). Mouse uterine NK cells are typically gated as lin-NK1.1+NKp46+ and comprise two main subsets, distinguished on the basis of CD49a and CD49b expression (CD49b is often referred to as DX5). Recent studies have shown that uNK in both species exhibit further heterogeneity (described in more detail later). The right hand panel shows selected receptors expressed on uNK cells and whether the corresponding MHC ligands are expressed on human or murine trophoblast cells. Receptors that normally inhibit NK activity are underlined. Note that LILRB1 which is normally considered an inhibitory receptor, has been reported to function as an activating receptor in uNK cells by some authors (57, 58); conversely NKp44 may function as an inhibitory receptor in uNK (54), so is shown underlined. C2+HLA-C, indicates an HLA-C allele carrying a C2 epitope. Full details and original references describing expression of each ligand on trophoblast are available in Gaynor and Colucci (59).
Figure 4
Figure 4
ILC subsets identified by mass cytometry of first trimester decidual cells. (A) tSNE landscape of CD45+ CD3- CD19- CD14- HLA-DR- (Lin-) cryopreserved decidual cells stained by mass cytometry. tSNE is coloured by clusters identified by DensVM clustering. 11 of these clusters can be assigned to NK or ILC subsets. Cluster 3 is heterogeneous and cluster 4 appears to contain cells damaged by cryopreservation so these were excluded from subsequent analysis. (B) Phenotypic characterisation of selected clusters. Size of the circle is representative of the proportion of the cluster positive for that marker. Circles are coloured by intensity of staining for that marker. Intensities have been scaled by marker (ie within each column). Red corresponds to higher expression and grey to lower expression. Percentage of cells in the cluster staining for each marker is indicated by size of the circles, scaled as shown on the right of the figure. (C) Box plots show the proportions of designated clusters within the total CD45+ Lin- decidual compartment. Blue dots = cryopreserved samples, Black dots = fresh samples. [ Figure 4A adapted from Huhn et al. (39), where details of the antibody panel are described]. (D) Comparison of decidual NK and ILC clusters identified by single cell RNA sequencing (scRNAseq) (40) and by mass cytometry (Cytof) (39). Identity of each cluster is based on their profiles of RNA or protein marker expression respectively. Cytof clusters 10-13, correspond to scRNAseq cluster dNK1 and are distinguished by their KIR expression. Cytof clusters 5,8 are distinguished by NKp44/NKG2A expression and correspond to scRNAseq cluster dNK3, representing ieILC. Cytof clusters 3 and 4 identified in panel A appear heterogeneous and don’t match any scRNAseq clusters; identities are not established. ++ is > 75% positive staining of cells with antibody in Cytof; + is 25% to 75%; − is <25%.
Figure 5
Figure 5
Frequencies of NK and ILC populations in decidua compared with other human tissues. Pie charts showing the frequencies of NK and ILC subsets in decidua (N = 7) compared with normal and pathological human tissues determined by mass cytometry. All charts, with the exception of first trimester decidua come from Simoni et al. (n = 4-9 separate donors for each tissue type) (33). For first trimester decidua, Live CD45+CD3-CD19-CD14-HLA-DR- cells were gated on as the parent population [data from Huhn et al. (39)]. Specified subsets were then identified on a tSNE landscape based on phenotypic profiles and their proportion within this population calculated. Unassigned cells (grey in decidua) could not be confidently labelled as a particular subset based on the markers included in the CyTOF panel. For all other tissues, samples were first depleted of T and B cells and then gated on Live CD45+ FcϵR1α– CD14– CD19– CD123–CD34-CD5- cells. Gating strategies to identify each subset were then as follows: NK cells (purple) = CD94+/-CD127+/-CD56+CD103-; ieILC1-like (red) = CD94+/-CD127+/-CD56+CD103+; ILC2 (blue) = CD94-CD127+CRTH2+; ILC3 =CD94-CD127+CRTH2-. [Figure adapted from Simoni et al. and Huhn et al. (33, 39)].
Figure 6
Figure 6
dNK receptor repertoire diversity Frequencies of the most common NK cell phenotypes were determined by mass spectrometry in freshly isolated CD45+Lin−CD56+ decidual NK cells for three selected individuals based on combinations of 26 individual phenotypic markers expressed on each cell. The twenty most frequent phenotypes detected are shown for each donor Dec1,3,5. Each column represents a phenotype shared by a number of cells; boxes are coloured if the marker is expressed in that phenotype. Frequencies of each phenotype as a percentage of total CD45+Lin−CD56+ are displayed as a heat map at the bottom of the figure. Based on analysis of data from Huhn et al. (39).
Figure 7
Figure 7
Dynamic changes of NK and ILC1s in the mouse uterus before and after pregnancy (A) Gating strategy to identify uterine NK and ILC1 subsets. Cells isolated from whole uterus were analysed together. Representative 2D FACS plot is from gestational day 9.5 (gd 9.5) and gated on Live CD45+CD3−CD19−CD11blow/- NK1.1+NKp46+ cells. ILC1s (red line) are CD49a+ Eomes−, tissue resident NK (trNK, blue) are CD49a+Eomes+, conventional NK (cNK, gold) are CD49a− Eomes+. (B) Proportions of indicated uterine subsets at different stages of the reproductive cycle as a percentage of the total Live CD45+CD3−CD19−CD11blow/- NK1.1+NKp46+ cells. w = weeks of age, gd = gestational day [Figure adapted from Filipovic et al. (81)].
Figure 8
Figure 8
Ligation of activating KIR on uNK affects human trophoblast migration in vitro. (A) Cartoon showing a cross section of a microfluidics device to study effects of uNK supernatants on migration of extravillous trophoblast (EVT) . Primary human EVT isolated from first trimester placentas are stained with tracker dye (blue) and embedded in Matrigel in the central channel. Cells are tracked in real time as they move between two side channels. These side channels have medium constantly flowing through them and can be supplemented with other factors or supernatants conditioned by uNK cells to create a gradient. (B) Plot showing motility of EVT towards or away from a microchannel containing uNK-conditioned medium. Freshly isolated uNK were purified from decidua using microbeads and KIR2DS1 was activated by EB6 antibody cross-linking for 12 hours. Stimulation conditions are shown on x-axis. On average, EVT display increased motility towards a channel containing supernatant from uNK stimulated by KIR2DS1 cross-linking. This is reduced by the addition of neutralising anti-GM-CSF in the supernatant. Control is ligation with isotype matched irrelevant IgG. Motility is calculated by subtracting the number of cells migrating upstream towards the uNK supernatant channel by the number of cells migrating away and dividing this by the total number of cells, [Figure adapted from Abbas et al. (120)].
Figure 9
Figure 9
Characteristics of innate lymphoid cells identified in the human uterus by scRNAseq and mass cytometry. Typical frequencies of each ILC type are given as a percentage of total decidual CD45+HLADR −CD14−CD19−CD3− (Lin−) gate in freshly isolated cells (not cryopreserved). Frequencies in endometrium will be different. Transcription factor (TF) expression and cytokine responses following PMA/ionomycin stimulation are based on mass cytometry data of Huhn et al. (39). TFs and cytokines listed in bold, indicate stronger staining. For functional responses, blank = 0-10%, + = 10-25%, ++ = 25-50%, +++ = 50+% staining by mass cytometry.
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