Human Term Pregnancy Decidual NK Cells Generate Distinct Cytotoxic Responses Ricardo De Mendonça Vieira, Ava Meagher, Ângela C

Human Term Pregnancy Decidual NK Cells Generate Distinct Cytotoxic Responses Ricardo de Mendonça Vieira, Ava Meagher, Ângela C. Crespo, Sarika K. Kshirsagar, Vidya Iyer, Errol R. Norwitz, This information is current as Jack L. Strominger and Tamara Tilburgs of September 29, 2021. J Immunol published online 6 May 2020 http://www.jimmunol.org/content/early/2020/05/05/jimmun ol.1901435 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 6, 2020, doi:10.4049/jimmunol.1901435 The Journal of Immunology

Human Term Pregnancy Decidual NK Cells Generate Distinct Cytotoxic Responses

Ricardo de Mendonc¸a Vieira,*,1,2 Ava Meagher,*,2 Aˆ ngela C. Crespo,*,† Sarika K. Kshirsagar,* Vidya Iyer,‡,x Errol R. Norwitz,‡,x Jack L. Strominger,* and Tamara Tilburgs*,{,‖

Decidual NK cells (dNK) are the main lymphocyte population in early pregnancy decidual mucosa. Although dNK decrease during pregnancy, they remain present in decidual tissues at term. First trimester dNK facilitate trophoblast invasion, provide protection against infections, and were shown to have many differences in their expression of NKRs, cytokines, and cytolytic capacity compared with peripheral blood NK cells (pNK). However, only limited data are available on the phenotype and function of term pregnancy dNK. In this study, dNK from human term pregnancy decidua basalis and decidua parietalis tissues were compared with pNK and first trimester dNK. Profound differences were found, including: 1) term pregnancy dNK have an increased degranulation response Downloaded from to K562 and PMA/ionomycin but lower capacity to respond to human CMV–infected cells; 2) term pregnancy dNK are not skewed toward recognition of HLA-C, as was previously shown for first trimester dNK; and 3) protein and gene expression profiles identified multiple differences between pNK, first trimester, and term pregnancy dNK, suggesting term pregnancy dNK are a distinct type of NK cells. Understanding the role of dNK throughout pregnancy is of high clinical relevance for studies aiming to prevent placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/

ecidual NK cells (dNK) are a specialized type of tissue- (1–4). Studies investigating the role of human dNK have thus far resident NK cells found at the maternal–fetal interface mainly focused on the first trimester of pregnancy. These studies D and display many phenotypic, functional, and transcrip- have suggested that dNK in first trimester human pregnancy act as tional differences compared with peripheral blood NK cells (pNK) cells that facilitate implantation, trophoblast invasion, and immunity to infection, processes that are all of key importance for early placental development and pregnancy success (5–8). The pro- *Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, portion of dNK significantly decrease during the course of by guest on September 29, 2021 MA 02138; †Program in Cellular and Molecular Medicine, Boston Children’s Hospital, ‡ pregnancy, but dNK remain present in both decidua basalis Boston, MA 02115; Department of Obstetrics and Gynecology, Tufts Medical Center, Boston, MA 02111; xMother Infant Research Institute, Tufts Medical Center, Boston, (d.basalis; decidual tissue that lines the site of implantation) { MA 02111; Division of Immunobiology, Center for Inflammation and Tolerance, and decidua parietalis (d.parietalis; decidual tissue lining the Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and ‖Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 uterine cavity and connecting to fetal chorionic tissue) at human 1 term pregnancy (9–11). dNK were present in placental samples Current address: BioSystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal. after preeclampsia and had a similar phenotype compared with 2R.d.M.V. and A.M. contributed equally. dNK in healthy controls (12). However, only very limited data are ORCIDs: 0000-0001-9892-7004 (R.d.M.V.); 0000-0002-9663-4707 (A.M.); 0000- available on the functional properties, including the cytolytic re- 0003-2323-1664 (S.K.K.); 0000-0003-4643-1546 (E.R.N.); 0000-0003-2734- sponses of term pregnancy dNK. 6485 (T.T.). Despite the expression of an abundance of the cytolytic mole- Received for publication December 3, 2019. Accepted for publication April 14, 2020. cules perforin (PFN) and granzyme B (GZMB) by dNK (1, 13, 14), This work was supported by National Institute of Allergy and Infectious Diseases, first trimester dNK were shown to have limited cytotoxicity in National Institutes of Health Grant R01-AI145862 and Strominger laboratory departmental response to classical MHC-negative target cells because of their grants. failure to polarize their cytotoxic granules to immune synapses Conceptualization: Aˆ .C.C., J.L.S., and T.T. Methodology: R.d.M.V., A.M., S.K.K., Aˆ .C.C., and T.T. Formal analysis: R.d.M.V., A.M., and T.T. Investigation: R.d.M.V., with these target cells (2). Furthermore, in vitro coculture of pri- A.M., and S.K.K. Resources: V.I. and E.R.N. Writing, original draft: R.d.M.V. and mary dNK and extravillous trophoblasts (EVT) obtained from the ˆ T.T. Writing, review, and editing: A.C.C., A.M., S.K.K., J.L.S., and T.T. Funding same pregnancy sample demonstrated an abundance of contacts acquisition: J.L.S. and T.T. Visualization: R.d.M.V. and T.T. Supervision: T.T. formed between EVT and dNK. In the contacts between dNK and Address correspondence and reprint requests to Dr. Tamara Tilburgs, Division of Immunobiology, Center for Inflammation and Tolerance, Cincinnati Children’s Hos- EVT, PFN did not localize to the immune synapse, and both dNK pital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: and pNK were unable to kill EVT, even when activated by proin- [email protected] flammatory cytokines (15). Furthermore, whereas dNK were shown to The online version of this article contains supplemental material. produce high levels of cytokines and growth factors (e.g., IL-8, Abbreviations used in this article: d.basalis, decidua basalis; dNK, decidual NK vascular endothelial growth factor [VEGF], and GM-CSF) in cell; d.parietalis, decidua parietalis; DSC, decidual stromal cell; EVT, extravillous trophoblast; FC, fold change; GNLY, granulysin; GZMB, granzyme B; HCMV, response to MHC-negative target cells (6, 16) these cytokines were + human CMV; KIR, killer Ig-like receptor; LILRB, leukocyte Ig-like receptor B; not produced in response to HLA-G EVT (17). MFI, mean fluorescence intensity; PFN, perforin; PMA/I, PMA/ionomycin; pNK, More recently, our laboratory and others have shown the ability peripheral blood NK cell; QPCR, quantitative PCR. of first trimester dNK to clear human CMV (HCMV)–infected cells Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 (5, 15). However, dNK only increased degranulation in response to

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901435 2 TERM PREGNANCY dNK ARE A DISTINCT TYPE OF NK CELLS

HCMV-infected maternal decidual stromal cells (DSC) and failed (e.g., d.parietalis versus d.basalis). Failure to regulate these dNK to increase degranulation and cytokine production in response to functions may contribute to exacerbation of placental inflam- HCMV-infected EVT (14). Thus, invading EVT of fetal origin seem mation, weakening of the fetal membranes, preterm rupture of protected from a dNK response, even when infected. This demon- membranes, and preterm delivery as well as increased maternal- strates the difficulty maternal immune cells face in providing im- to-fetal transmission of pathogens. Thus, detailed investigation of munity to infection in the tolerogenic environment of the placenta. how dNK types and/or dNK functions change throughout preg- dNK but not pNK also constitutively secrete high levels of the an- nancy is essential for understanding development of pregnancy tibacterial peptide granulysin (GNLY) (18). The 15-kDa GNLY complications. propeptide was shown to act as an alarmin involved in leukocyte recruitment, whereas the processed 9-kDa isoform of GNLY binds and disrupt cholesterol-poor membranes (i.e., bacterial, fungal, and Materials and Methods parasitic membranes) and enhance clearance of these infections (19). Blood and tissue samples Thus, dNK may use degranulation-independent mechanisms to Collection of blood and tissue samples has recently been described, and its provide immunity to placental infection while maintaining im- description in this study is adapted from Refs. 34 and 35. Discarded human mune tolerance to invading trophoblasts. placental and decidual materials (gestational age 6–12 wk) were obtained from women undergoing elective pregnancy termination at a local repro- HLA-C is the only polymorphic MHC molecule expressed by ductive health clinic. Term placental tissues (gestational age .37 wk) were EVT that can be recognized by maternal allogeneic T cells and NK obtained from healthy women after uncomplicated pregnancy at term cells (20, 21). dNK in first trimester were shown to have increased delivered by elective cesarean section or uncomplicated spontaneous expression of the HLA-C–binding killer Ig-like receptors (KIRs). vaginal delivery at Tufts Medical Center. No pregnancies from which term Downloaded from placentas were collected had clinical evidence of chorioamnionitis (intra- This includes the inhibitory KIR2DL1 and KIR2DL2/3, which amniotic infection). Furthermore, all tissues were visually inspected for recognize HLA-C2 and HLA-C1 group allotypes, respectively, as signs of excessive inflammation (including discoloration, large infarctions, well as the activating receptor KIR2DS1, which recognizes HLA-C2 and foul odor) and only healthy tissues were used for further processing. group allotypes (14, 16, 22, 23). It was shown that women who lack Peripheral blood leukocytes, including pNK, were isolated from discarded KIR2DS1 in their genome while carrying a fetus with an HLA-C2 leukopacks from healthy nonpregnant volunteer blood donors at the Massachusetts General Hospital in Boston, MA. All human tissue used for group allotype are at increased risk of developing pregnancy com- this research was de-identified, discarded clinical material. No clinical http://www.jimmunol.org/ plications (16, 24). The activation through HLA-C2/KIR2DS1 was information, including the fetal sex and sex of blood donors, was available postulated to activate dNK to secrete cytokines and growth factors, for analysis. The Committee on the Use of Human Subjects (the Harvard especially GM-CSF, to facilitate trophoblast invasion and placental Institutional Review Board) determined that this use of placental and decidual material is not-human-subjects research. Term placental tissue was growth (16). More recently, our laboratory provided evidence that collected under a protocol approved by Tufts Health Sciences Institutional the expression of KIR2DS1 by dNK increases the ability of dNK but Review Board. not pNK to respond to HCMV-infected DSC (14). The enhanced ability of KIR2DS1+ dNK to control placental HCMV infection may Isolation of pNK and dNK limit subsequent virus-induced placental pathology. Isolation of pNK and dNK has recently been described, and its description in The recognition of polymorphic HLA-C allotypes by dNK this study is adapted from Refs. 14 and 17. In short, first trimester villous by guest on September 29, 2021 from distinct maternal KIR haplotype backgrounds may lead to and decidual tissues from elective pregnancy terminations were macroscopically identified and separated. d.parietalis from term pregnancy was alloincompatibility. However, a multitude of additional NK cell collected by removing the amnion and delicately scraping the d.parietalis receptors and ligands expressed by EVT contribute to inhibition from the chorion. d.basalis was macroscopically dissected from the ma- and activation of dNK activity (25). Particularly, the interaction of ternal side of the placenta. Collected decidual tissues were washed with nonpolymorphic HLA-E and HLA-G molecules with the HLA-E PBS, minced, and thereafter digested with 0.1% collagenase type IV and 0.01% DNase I (Sigma-Aldrich), shaking at 37˚C for 1 h 15 min. Resulting receptors CD94/NKG2 and the HLA-G receptor KIR2DL4 was decidual lymphocyte suspensions were washed with RPMI 1640 (Life shown to limit NK cell cytotoxicity and induce IFN-g secretion Technologies) containing 10% FBS (Atlanta Biologicals) for 8 min at (26). The HLA-G receptors leukocyte Ig-like receptor B (LILRB) 1800 rpm and filtered through 100-, 70-, and 40-mm sieves (BD Labware, 1 and LILRB2 have an inhibitory motif and lead to inhibition of Franklin Lakes, NJ). Lymphocytes were dissolved in 20 ml 1.023 g/ml NK cells upon binding (27). Other NKRs (e.g., NKp30, NKp44, Percoll (GE Healthcare) and layered on a Percoll gradient (10 ml 1.080 g/ml; 15 ml 1.053 g/ml). After density gradient centrifugation (25 min, NKp46), coreceptors (e.g., DNAM1 and 2B4), and receptors for 2000 rpm), decidual lymphocytes were isolated from the 1.080–1.053 g/ml coinhibitory molecules (e.g., T cell immunoreceptor with Ig and interface, washed twice with RPMI 1640, and directly stained for flow ITIM domains [TIGIT] and programmed cell death protein 1 [PD-1]) cytometric analysis (BD LSR II) or FACS sort (BD FACSAria II). pNK from may provide further ways to differentially modulate pNK and healthy nonpregnant blood donors were isolated using a RosetteSep Human NK Cell Enrichment Cocktail (STEMCELL Technologies) followed by Ficoll dNK function and may contribute to distinct pNK and dNK (GE Healthcare) density gradient centrifugation (20 min, 2000 rpm). subtypes (28, 29). This diversity of NKRs combined with genetic and environmental determinants has identified an estimated 6000– Isolation of EVT 30,000 phenotypes of human NK cell populations within an in- Isolation of EVT has been recently described and its description in this study dividual (30). A recent study using single-cell RNA-sequencing was adapted from Refs. 17 and 34. In short, first trimester villi were gently technologies of the early maternal–fetal interface in humans identified scraped from the basal membrane and digested with trypsin (0.2%)–EDTA three dNK states (31). During murine gestation, both conventional NK (0.02%) for 8 min at 37˚C. Cells were washed with DMEM/F12 medium containing 10% newborn calf serum and 1% penicillin/streptomycin (all cells and tissue-resident NK cells were shown to accumulate in both from Life Technologies) and filtered over a gauze mesh. The filtrate was the d.basalis and myometrium (32, 33). washed again and layered on Ficoll (GE Healthcare) for density gradient Combined, these data suggest that beyond the vast NK cell centrifugation (20 min, 2000 rpm). Cells were collected, washed, and in- diversity based on NKR expression, distinct NK cell types or cubated for 20 min at 37˚C in a tissue culture plate for removal of macrophages. Cells were harvested for Ab staining and flow cytometry. differentiation states exist that can accommodate a variety of dNK functions at the human maternal–fetal interface. These include Cell culture and NK cell degranulation assays secretion of cytokines and growth factors, interaction with EVT, Cell culture and NK cell degranulation assays have recently been described allorecognition, and immunity to infection. These functions may and its description in this study is adapted from Refs. 14 and 35. pNK and change depending on gestational age and decidual tissue type dNK were cultured in X-VIVO 10 Media with gentamicin (Lonza), 5% The Journal of Immunology 3 human AB serum (Corning), and 2.5 ng/ml IL-15 (BioLegend). DSC were (American Type Culture Collection) and collecting supernatants. Supernatants cultured in Opti-MEM (Life Technologies) supplemented with 3% FCS were snap frozen in liquid nitrogen until use. DSC at 80% confluence in and 25 mg/ml gentamicin and passaged once to twice a week (36). K562 48 wells were infected at a multiplicity of infection of 0.5 for 1 h at 37˚C, and JEG-3 were cultured in RPMI 1640 10% FCS. For degranulation as- followed by removal of the inoculum and addition of fresh media. For says, dNK and pNK were cultured overnight in X-VIVO 10 Media with 2.5 coculture assays HCMV-infected cells were washed after 24 h of infection, ng/ml IL-15 (BioLegend). The dNK and pNK (effectors) were counted and and 75,000 pNK or dNK were added. cocultured without or with 2.5 ng/ml PMA in combination with 0.1 mg/ml ionomycin or K562 in a 1:3 E:T ratio in 96-well plates for 2 h. dNK and RNA isolation and quantitative PCR chip analysis pNK were also cocultured with uninfected and HCMV-infected DSC. RNA isolation and quantitative PCR (QPCR) chip analysis has recently DSC were typed for HLA-C at the American Red Cross (Dedham, MA) been described, and its description in this study is adapted from Ref. 34. and expressed both HLA-C1 and HLA-C2. Seventy-five thousand + 2 + CD45 CD14 CD56 NK cells from blood, decidua (6–12 wk), d.basalis freshly isolated dNK or pNK were added to confluent healthy uninfected and d.parietalis (.37 wk) were purified by FACS sort and collected directly DSC or HCMV-infected DSC, plated in 48-well plates in X-VIVO 10 into 600 ml of TRIzol reagent (Life Technologies) and stored at 280˚C until Media supplemented with 2.5 ng/ml IL-15 for 16 h. Two hundred use. RNA was isolated using the miRNAeasy Micro Kit (QIAGEN) per and fifty nanograms per milliliter CD107a PerCP–Cy5.5 Ab was added manufacturer’s instruction. RNA was analyzed on a NanoDrop to determine to all cocultures. NK were collected and fixed for 10 min in 1% PFA RNA yield and integrity. The RNA quality of all samples was further con- and subsequently stained with all relevant surface markers for FACS firmed by performing a QPCR analysis for GAPDH. In short, RNA was analysis. reverse transcribed with Stratagene’s AffinityScript QPCR. A cDNA Syn- Flow cytometry thesis Kit and amplification of specific PCR products for GAPDH was detected using the SYBR Green system (Applied Biosystems) in triplicates. Flow Cytometry has recently been described and its description in this Subsequently, high-quality samples were run in duplicate on the BioMark study is adapted from Refs. 14, 34, and 35. Abs used for flow cytometric Fluidigm QPCR 96.96 chip according to manufactures instructions. Briefly, analysis and FACS sorting are listed in Table I. For surface staining, cells DNA was preamplified in a 96-well plate using the Fluidigm PreAmp Master Downloaded from were stained for 30 min on ice in PBS with 1% FCS. For KIR2DS1 Mix combined with forward and reverse primers of each gene. Exonuclease staining, NK were stained with KIR2DL1 first (clone 143211, 1.5 mg/ml, treatment to remove unincorporated primers was carried out using Exonu- 20 min) and thereafter with KIR2DL1/S1 (clone EB6, 0.5 ml, 15 min) as clease I at 40 U/ml (New England BioLabs) for 30 min at 37˚C and 15 min described previously (14). For intracellular staining, cells were fixed and 80˚C. A 5-fold dilution was prepared in TE BUFFER (PN T0224; Teknova). permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences). The sample premix was prepared using with a 203 DNA binding dye Analysis was performed on a FACSCalibur (BD Biosciences) or a BD sample loading reagent (Fluidigm) and 23 SsoFast EvaGreen supermix with

LSR II (BD Biosciences), and the FACS sort was done using a BD low ROX (Bio-Rad Laboratories). The assay mix was prepared in a separate http://www.jimmunol.org/ FACSAria III. 96-well plate consisting of 23 assay loading reagent, 13 DNA suspension Infection of DSC buffer, and 100 mM mixed forward and reverse primers. Chips were primed, loaded with both assay and sample mix, and run on BioMark readout in- Infection of DSC has recently been described, and its description in this struments as described by the manufacturer. BioMark data were processed study is adapted from Refs. 14 and 34. High-titer virus stocks of HCMV– using Fluidigm BioMark software. Quality control was based on the AD169 (IE-1-GFP) were obtained by infecting human foreskin ﬁbroblasts EvaGreen reagent allowing for detection of unspeciﬁc PCR product and

Table I. List of Abs by guest on September 29, 2021 Ab Conjugate Clone Source 2B4 Pacific Blue C1.7 BioLegend CD3 PerCP UCTH1 BioLegend CD14 PerCP HCD14 BioLegend CD45 PerCP; allophycocyanin HI30 BioLegend CD45 Pacific Orange HI30 Thermo Fisher Scientific CD56 Alexa Fluor 700, PE, Pacific Blue NCAM BioLegend CD57 Pacific Blue HNK-1 BioLegend CD94 PE/Cy7 DX22 BioLegend CD107a PerCP-Cy5.5 H4A3 BioLegend DNAM1 FITC 11A8 BioLegend EGFR1 FITC AY13 BioLegend GNLY Alexa Fluor 488 RB1 BD Biosciences GNLY PE DH2 BioLegend GZMB PE/Texas Red GB11 Thermo Fisher Scientific HLA-C PE DT-9 BD Biosciences HLA-E PE 3D12 eBioscience HLA-G PE MEM/G9 Abcam IgG1 FITC, Alexa Fluor 488, PE, allophycocyanin, MOPC-21 BioLegend PerCP/Cy5.5, PE/Cy7, Pacific Blue IgG2a PE, PE/Cy7, Alexa Fluor 647 MOPC-173 BioLegend IgG2b Pacific Blue, PerCP/Cy5.5 MPC-11 BioLegend KIR2DL1 Allophycocyanin 143211 R&D Systems KIR2DL1/S1 FITC HP-MA4 BioLegend KIR2DL2/3 PE CH-L BD Biosciences KIR2DS1 PE/Cy7 EB6B Beckman Coulter KIR2DL4 Allophycocyanin mAb33 BioLegend LILRB1 PerCP/Cy5.5 GHI/75 BioLegend LILRB2 PE/Cy7 42D1 BioLegend MICA/B Alexa Fluor 647 6D4 BioLegend NKG2A PE 131411 R&D Systems NKG2C PE 134591 R&D Systems NKG2D Allophycocyanin 1D11 BioLegend NKp30 Allophycocyanin P30-15 BioLegend NKp46 FITC 9E2 BioLegend PFN Pacific Blue dG9 BioLegend 4 TERM PREGNANCY dNK ARE A DISTINCT TYPE OF NK CELLS formation of primer dimers using the BioMark software. Samples of Expression of cytolytic molecules in dNK varies failed reactions were automatically excluded by the software. The throughout pregnancy resulting values for 34 genes of 32 samples were normalized against GAPDH (DCT), and the fold change (FC) of relative gene expression Freshly isolated pNK and dNK were examined for intracellular was calculated relative to the median DCT of each gene. K-means expression of the cytolytic molecules GZMB, PFN, total GNLYand cluster analysis was performed using the FC values of the 34 genes the 9-kDa–active form of GNLY. Although close to 100% of all in ExpressCluster v1.3 in GenePattern (https://cloud.genepattern.org/gp). Differential expression of significantly correlating genes was based on pNK and dNK in first trimester and term pregnancy expressed K-means with a minimum of a 2-FC. PFN, GZMB, and GNLY (Fig. 2A, 2B), the mean fluorescence intensity (MFI) of PFN and GZMB expression was significantly Statistical analyses higher in pNK compared with dNK of all groups (Fig. 2C). No All data were analyzed using GraphPad Prism version 6.07 software. To significant differences were found in PFN and GZMB expression determine differences between two paired groups, a nonparametric Wil- in dNK in first trimester and term pregnancy (Fig. 2A, 2B). Ex- coxon signed-rank test was performed (Fig. 5B). To determine differences pression levels (MFI) of total GNLY and 9-kDa GNLY as well as among more than two unpaired groups, a nonparametric Kruskal–Wallis + test (without matching or pairing) with a Dunn multiple comparison the proportion of 9-kDa GNLY cells was significantly higher in posttest was performed. For the Dunn posttest, the mean ranks were first trimester dNK compared with pNK and dNK from term compared with the mean rank of every column (Figs. 1B, 2B, 2C, pregnancy decidual tissues (Fig. 2). Thus, whereas close to 100% 3B) or control column A (Fig. 4C, Supplemental Figs. 1A, 1B, 2C, of dNK throughout pregnancy express all cytolytic granules (PFN, 3B, 3C) or control column B (Fig. 6). The p values ,0.05 were considered to denote significant differences. The p values (*p , 0.05, GZMB, and GNLY), the expression of GNLY and 9-kDa GNLY **p , 0.01, ***p , 0.005) are indicated within each figure. Sample size by dNK varies and has a noticeable drop at term pregnancy indicates biological replicates of individual placental of blood NK cell compared with first trimester pregnancy. Downloaded from isolates and are indicated in each figure legend. Sample sizes were ND beforehand. Term pregnancy dNK have distinct cytolytic capacity compared with pNK and first trimester dNK Results First, degranulation, as a measure for cytotoxic activity of pNK The proportion of dNK decreases toward term pregnancy but and dNK in response to MHC-negative K562 targets cells and dNK remain present PMA/ionomycin (PMA/I) stimulation, was determined using the http://www.jimmunol.org/ FACS analysis on freshly isolated pNK from healthy nonpreg- CD107a-dependent degranulation assay. pNK and dNK were nant blood donors and dNK isolated from first trimester decidua cultured overnight with a low dose of 2.5 ng/ml IL-15. The pNK (gestational age 6–12 wk), and term placenta d.basalis and and dNK were counted and cocultured without or with PMA/I or d.parietalis (gestational age .37 wk) was performed to deter- K562, and degranulation was determined using CD107a as a mine the proportions of CD45+CD142CD32CD56+ pNK and marker. As reported previously (2), the response of first trimester dNK (Fig. 1A, Table I). The proportion of dNK among CD45+ dNK to K562 and PMA/I was significantly lower than that of pNK cells in first trimester decidua (median = 66%, range = 36–84%) (Fig. 3). However, degranulation of term pregnancy dNK from was significantly higher than the proportion of pNK (median = 8%, both d.basalis and d.parietalis in response to K562 and PMA/I was by guest on September 29, 2021 range = 3–15%) and dNK in term pregnancy d.basalis (median = 24%, significantly higher than that of first trimester dNK and compa- range = 12–65%) and d.parietalis (median = 30%, range = 10–49%) rable to the pNK responses (Fig. 3A). Interestingly, IFN-g and (Fig. 1B). No significant correlation was observed between the TNF-a production in response to PMA/I of both first trimester and percentage of CD56+ NK cells in paired samples of d.basalis term pregnancy dNK was comparable and both were significantly and d.parietalis (Fig. 1C). Thus, whereas the proportion of dNK lower than the IFN-g and TNF-a production by pNK (Fig. 4). decreased at term pregnancy, dNK remained present and ele- Next, the capacity of pNK and dNK to degranulate in response to vated in decidua compared with blood throughout pregnancy. A HCMV-infected DSC compared with healthy uninfected DSC limitation of this study is the lack of clinical information on the was determined as described previously (14). Whereas virtually blood and tissues used for experiments that may have an impact all pNK as well as dNK from first trimester decidua had an in- on NK cell phenotypes and contribute to the variation in the NK creased level of degranulation in response to HCMV-infected cell proportions observed in this study. DSC compared with healthy uninfected DSC, only four out of

FIGURE 1. The proportion of dNK decreases toward term pregnancy, but dNK remain present. (A and B)FACSgatingstrategytoselect CD45+CD32CD142CD56+ NK cells in blood, decidua at 6–12 wk, d.basalis .37 wk, and d.parietalis .37 wk. (C) Graph depicts percentages of CD45+CD32CD142CD56+ NK cells of CD45+ lymphocytes in blood, decidua at 6–12 wk, d.basalis .37 wk, and d.parietalis .37 wk. (D)No signiﬁcant correlation was observed between the percentages of CD56+ NK cells in paired samples of d.basalis and d.parietalis (n = 12–-18). Bars represent median and interquartile range *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5

FIGURE 2. Expression of cytolytic molecules in pNK and dNK. (A) Representative FACS plots of intracellular expression of PFN, GZMB, GNLY, and 9-kDa GNLY compared with isotype controls in pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk. Graphs depict percentage (B)andMFI(C)ofPFN,GZMB, Downloaded from GNLY, and 9-kDa GNLY expression in all NK types. Bars represent median and interquartile range (n = 11–16). **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ by guest on September 29, 2021

eight term pregnancy dNK increased their response to HCMV- toward recognition of HLA-C. Next, the cell surface expression of infected DSC (Fig. 5A, 5B). Furthermore, the FC in degranula- the inhibitory HLA-E receptor NKG2A, the activating HLA-E tion in response to HCMV-infected relative to healthy uninfected receptor NKG2C, the MICA/B–activating receptor NKG2D, and DSC was calculated and was significantly decreased in term the coreceptor CD94 were determined by flow cytometry on pNK pregnancy dNK compared with pNK (Fig. 5C). First trimester and dNK. Whereas dNK expressed significantly higher CD94, dNK had a small but NS reduction in the relative degranulation NKG2A, and NKG2C compared with pNK, no significant dif- compared with pNK. Thus, whereas term pregnancy dNK have ferences in CD94 and NKG2A expression between the three dNK increased capacity to respond to K562 cells and PMA/I stim- types were observed (Supplemental Fig. 1B, 1C). Interestingly, the ulation, their cytolytic response to HCMV-infected DSC is expression of NKG2D was increased on term pregnancy dNK reduced. from both d.basalis and d.parietalis compared with pNK and first trimester dNK, suggesting the capacity to clear MICA/B–positive Expression of NKR by first trimester and term pregnancy dNK cells increases toward term pregnancy. Next, expression of the contain key differences HLA-G receptors KIR2DL4, LILBR1, and LILRB2 were detected The difference in cytolytic capacity between first trimester and term by QPCR on RNA isolated from all NK cell types. KIR2DL4 pregnancy dNK suggest these cells may have differences in the was highly expressed by first trimester and term pregnancy expression of NK cell receptors involved in their activation and dNK but not pNK (Fig. 6B, left panel). LILRB1 was highly inhibition. First, cell surface expression of the inhibitory HLA-C expressed by first trimester dNK and pNK but not by term receptors KIR2DL1 and KIR2DL2/3 and the activating HLA-C pregnancy dNK (Fig. 6B, middle panel), whereas LILRB2 was receptor KIR2DS1 was determined by flow cytometry on pNK highly expressed by first trimester dNK and term pregnancy and dNK, as described previously (14). Although KIR2DL1, dNK from d.parietalis only (Fig. 6B, right panel). QPCR data KIR2DL2/3 and KIR2DS1 were significantly increased on first for KIR2DL4, LILBR1, and LILRB2 expression was confirmed trimester dNK compared with pNK as reported previously (22), by flow cytometry (Fig. 6C). Lastly, the cell surface expression of term pregnancy dNK from both d.basalis and d.parietalis did not the NKRs NKp30 and NKp46, the coreceptors 2B4 and DNAM1, have increased KIR2DL1, KIR2DL2/3, and KIR2DS1 expression, and the NK maturation marker CD57 were determined by flow and their expression was comparable to pNK (Fig. 6A, Supplemental cytometry. NKp30 expression was low on all NK cells analyzed, Fig. 1A). Thus, unlike dNK in the first trimester, term preg- with no significant differences between pNK and dNK types nancy dNK have fewer HLA-C receptors and dNK are not skewed (Supplemental Fig. 2A, 2B). Although nearly 100% of all NK 6 TERM PREGNANCY dNK ARE A DISTINCT TYPE OF NK CELLS

FIGURE 3. Term pregnancy dNK have a distinct capacity to degranulate in response to PMA/I and K562. (A) Representative FACS plots of CD56 and CD107a staining in pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk cultured alone (top panels) and with PMA/I (middle panels) and K562 (bottom panels). (B) Percentage of CD107a+ NK cells from blood, decidua at 6–12 wk, d.basalis .37 wk, and d.parietalis .37 wk upon being cultured alone (left), Downloaded from with PMA/I (middle), and K562 (right). All cells were cultured in the presence of 2.5 ng/ml IL-15 for 2 h (n = 6–16). Bars represent median and interquartile range. *p , 0.05, **p , 0.01, ***p , 0.005. http://www.jimmunol.org/ by guest on September 29, 2021 types expressed NKp46, the MFI of NKp46 expression was NKRs and coreceptors that might facilitate NK cell activation signiﬁcantly higher in all dNK types compared with pNK and immune synapse formation with different target cells, (Supplemental Fig. 2A, 2B). In contrast, the expression of including EVT and virus-infected cells.

CD57 was exclusively found on pNK, whereas the expression + of 2B4 and DNAM1 was signiﬁcantly higher on pNK com- HLA-G EVT in ﬁrst trimester and term pregnancy and pared with all dNK (Supplemental Fig. 2A, 2C). These data HCMV-infected DSC offer distinct ligands for dNK receptors highlight that pNK and the three types of dNK analyzed in this First, healthy DSC and HCMV-infected DSC as described above study contain profound differences in their expression of were analyzed for the expression of HLA-C, HLA-E, and MICA/B.

FIGURE 4. IFN-g and TNF-a production by pNK and dNK in response to PMA/I. (A) Repre- sentative histograms of intracellular IFN-g and TNF-a staining and an IgG control in CD56+ pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk cultured with PMA/I. (B) Percentage of IFN-g+ and TNF-a+ NK cells and (C) MFI of IFN-g+ and TNF-a+ NK cells from blood, decidua at 6–12 wk, d.basalis .37 wk, and d.parietalis .37 wk upon PMA/I stimulation. All cells were cultured in the presence of 2.5 ng/ml IL-15 for 2 h (n = 6). Bars represent median and interquartile range. *p , 0.05, **p , 0.01. The Journal of Immunology 7

FIGURE 5. Term pregnancy dNK have decreased response to HCMV-infected DSC. (A) Representative FACS plots of CD56 and CD107a staining in pNK and dNK cultured alone (top panels), with healthy uninfected DSC (middle panels), and with HCMV-Infected DSC (bottom panels). (B) Percentage of CD107a+ pNK, dNK from 6-to-12 wk decidua, d.basalis .37 wk, and d.parietalis .37 wk combined after coculture with healthy/HCMV-free (2) and HCMV-infected (+) DSC. (C) FC in degranulation of pNK and dNK in Downloaded from response to HCMV-infected cells relative to healthy cells are depicted. All ratios .1 indicate an increased response to HCMV-infected versus uninfected cells. All cells were cultured in the presence of 2.5 ng/ml IL-15 for 16–18 h (n = 8–13). Bars represent median and interquartile range. ,

**p 0.01. http://www.jimmunol.org/ by guest on September 29, 2021

Although HLA-E and MICA/B expression did not change upon each gene. K-means cluster analysis based on a minimum 2-fold HCMV infection, HCMV infection downregulated HLA-C on difference of gene expression in the four NK cell populations approximately half of the infected DSC (Fig. 7A). Thus, HCMV visualized four separate gene clusters. Cluster I identified a set infection of DSC may render these cells more susceptible to killing of genes, including EOMES, GATA3, GNLY, GZMK, CD56, through missing self-recognition involving KIR2DL1/2/3. The ITGA1, and KIR2DL1, that were highly expressed by first other half of DSC still expressing HLA-C upon HCMV infec- trimester dNK and reduced in pNK and term pregnancy dNK tion may facilitate interactions of the activating HLA-C receptor (Fig. 8A). Cluster II identified a set of genes, including TBX21, KIR2DS1 expressed by dNK. Furthermore, EVT were isolated TNF, NKp44, CSF2, and VEGFA, that are upregulated in pNK from first trimester villi at 6–12 wk, term placental villi at compared with all dNK (Fig. 8B). Cluster III identified a set of .37 wk, and chorionic tissue at .37 wk. FACS analysis dem- genes, including IFN-g, GZMH, IFNGR1, PDCD1, and KLRG1, onstrated no significant differences in expression of HLA-C and that are upregulated by term pregnancy dNK from both d.basalis HLA-E on these cells. In contrast, HLA-G expression was in- and d.parietalis, compared with first trimester dNK and pNK creased on the term placenta EVTs, providing increased in- (Fig. 8C). Cluster IV identified a set of genes, including PRF1, teraction through KIR2DL4 and LILRB2 expressed by term CD69, ITGB2, and NKp80, that are upregulated by term preg- pregnancy dNK (Fig. 7B). nancy dNK from d.parietalis compared with first trimester dNK and pNK (Fig. 8D). The increase in PFN mRNA in this study did mRNA expression profiles reveal significant differences not correspond with the PFN protein levels observed (Fig. 2). between pNK and all dNK types Previously, we demonstrated that decidual CD8+ Tcellshad To further investigate the function and the molecular mechanisms reduced PFN and GZMB protein and increased mRNA expres- pNK, first trimester dNK, and term pregnancy dNK use to interact sion and speculated that microRNAs may play a role in regu- with target cells, RNA was isolated from CD45+CD142CD32CD56+ lation PFN and GZMB expression (35, 37). Other genes NK cells purified from blood, decidua at 6–12 wk, d.basalis .37 wk, included on the gene expression assay that were not differen- and d.parietalis .37 wk. The BioMark Fluidigm QPCR chip was tially expressed include LGALS1, NKG2E, FASLG, CD25, and used to detect gene expression for a panel of NKRs, cytolytic TIM3, and genes that were not expressed or failed include IL-6, molecules, cell surface markers, cytokines, and transcription IL-8, IL-10, IL-17A, and IL-22. Bar graphs of all differentially factors. Thirty-five out of forty-eight primer pairs included on expressedgenesbetweenpNKanddNKaredepictedinSupplemental the chip had detectable CT values and melting curves. CT values Fig. 3, and an overview of all differences are depicted in were normalized against GAPDH (DCT), and the FC of relative Supplemental Table I. These data further support that pNK and gene expression was calculated relative to the median DCT of the three types of dNK analyzed in this study display profound 8 TERM PREGNANCY dNK ARE A DISTINCT TYPE OF NK CELLS

FIGURE 6. Differential expression of HLA-C and HLA-G receptors by pNK and dNK. (A) Graphs depict percentage of KIR2DL1+, KIR2DL2/3+,and KIR2DS1+ cells within freshly isolated pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk. The graph depicting the percentage of KIR2DS1+ NK cells only contains +

samples with a clear KIR2DS1 NK population Downloaded from (Supplemental Fig. 1A). (B) Graphs depict mRNA expression of KIR2DL4, LILRB1, and LILRB2 relative to GAPDH in freshly isolated pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d. parietalis .37 wk (n = 4–8). Bars represent median and interquartile range. *p , 0.05, **p , 0.01,

***p , 0.001. (C) Representative FACS histograms http://www.jimmunol.org/ of intracellular (IC) KIR2DL4 and cell surface LILRB1 and LILRB2 expression on freshly isolated pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk. Representative images of three biological isolates are shown. by guest on September 29, 2021

differences and suggest term pregnancy dNK are a separate type dNK compared with pNK, whereas term pregnancy dNK had of NK cells. increased responses compared with first trimester dNK. This suggests that term pregnancy dNK are a functionally distinct Discussion type of NK cell. In addition, the capacity of first trimester dNK Numerous gaps remain in our understanding of whether and how to degranulate in response to HCMV-infected cells was not de- dNK interact with EVT to facilitate EVT invasion and promote creased compared with pNK, whereas term pregnancy dNK had placental development while simultaneously providing protective equal degranulation responses to PMA/I and K562 compared immunity for a variety of pathogens. In this study, analysis of with pNK but a reduced capacity to respond to HCMV-infected dNK throughout human pregnancy identified key differences in DSC. Overall, these data should revisit the current narrative dNK function, phenotype, and gene expression signatures be- around dNK cytotoxicity and take into account their tissue origin tween dNK purified from the first trimester and term pregnancy, and gestational age as well as the target cells or stimulation and some additional differences were observed between dNK protocols used to determine their cytotoxicity. Most importantly, purified from d.basalis and d.parietalis tissues. further understanding of dNK requires additional investigation into Based on first trimester data, dNK have previously been distinct dNK subtypes with possible defined functions for immunity considered low cytotoxic (2). Whereas it is true that fewer first and tissue development. Presence of dNK types that interact with trimester dNK degranulate in response to PMA/I or MHC- EVT to facilitate their invasion and placental development are likely negative target cells, such as K562 cells, over one third of the to be increased in first trimester decidua, when trophoblast invasion first trimester dNK did respond to these stimuli (compared with is most active. These dNK types would also more likely be found in two thirds of pNK). Although degranulation and cytokine pro- d.basalis, the site of implantation, compared with d.parietalis, the duction were not analyzed simultaneously in the experiments placental membranes where d.parietalis connects to chorionic performed in this study, degranulation, IFN-g,andTNF-a pro- HLA-G+ trophoblasts that have more limited capacity for invasion duction in response to PMA/I were all decreased in first trimester of uterine tissues. In contrast, dNK types responsible for immunity The Journal of Immunology 9 Downloaded from

FIGURE 8. Gene expression profiles of pNK and dNK. K-means clus- tering identified four clusters of genes that significantly correlate their mRNA expression pattern between pNK and dNK from 6-to-12-wk decidua, d.basalis .37 wk, and d.parietalis .37 wk. (A) Cluster I identified http://www.jimmunol.org/ genes highly expressed by dNK from 6-to-12-wk decidua. (B) Cluster II identified genes highly expressed by pNK. (C) Cluster III identified genes . . D + highly expressed by dNK d.basalis 37 wk and d.parietalis 37 wk, and ( ) FIGURE 7. Differential NKR ligand expression by HLA-G EVT and cluster IV identified genes highly expressed by dNK in d.parietalis .37 wk. A HCMV-infected DSC. ( ) Representative FACS histograms of HLA-C, Mean relative expression values (n = 8) for all NK cell types are depicted. HLA-E, and MICA/B expression in uninfected DSC and DSC infected with HCMV compared with IgG control. (B) Representative FACS histograms of HLA-C, HLA-E, and HLA-G expression in freshly isolated HLA- G+ EVT 6–12 wk, isolated HLA-G+ EVT .37 wk, and chorionic HLA-G+ Additionally, other reports addressing dNK subtypes identified + + by guest on September 29, 2021 EVT .37 wk compared with isotype controls. Representative images of EOMES CD49a cells in the virgin mouse uterus and in early three biological isolates are shown. pregnancy murine decidual tissues (32, 38). These studies showed the presence of EOMES+CD49a+CD49b2 NK cells at gestational day 5.5, which mainly produced TNF and decreased to infection may play a role throughout pregnancy, but subtypes during gestation. Another population of EOMES+CD49a2CD49b+ may vary to accommodate distinct requirements for trophoblast NK cells, enriched in IFN-g producing cells, were predominant invasion and immune tolerance. during midgestation (gestational day 10.5) and were suggested to A recent study using single-cell RNA-sequencing technologies contribute to spiral artery remodeling. These reports were confirmed of the early maternal–fetal interface in humans did indeed identify by the observation in our present study: expression of EOMES and three dNK states (31). dNK1 were characterized by high expres- CD49a (also known as ITGA1) is significantly increased in human sion of KIR, NKG2Rs, cytolytic granules (PFN, GNLY, GZMA, first trimester dNK compared with pNK and term pregnancy dNK. and GZMB), and CSF1 expression; dNK2 were characterized by First trimester dNK also had a significant drop in IFN-g expression, expression of ITGB2, GZMH, CD27, and ANXA1, whereas dNK3 confirming their resemblance to the murine EOMES+CD49a+CD49b2 lacked KIR and NKG2Rs and had increased expression of CD160, NK population. Expression of TNF-a was highest in pNK and TIGIT, and CCL5. Our data clearly show increased proportions of term pregnancy d.basalis dNK and decreased in first trimester dNK dNK in the first trimester that resemble the dNK1 population. and term pregnancy d.parietalis dNK. Several studies have described First trimester dNK had high KIR and NKG2R expression and the importance of TGF-b and DSC in the differentiation of dNK, but an abundance of cytolytic granule proteins as measured by flow limited attention was given to distinct dNK subtypes (39, 40). cytometry and mRNA expression. Particularly striking is the Expression of EOMES in combination with TBX21 (also known very high expression of 9- and 15-kDa GNLY in first trimester as T-bet), both members of the T-box transcription factor family, dNK compared with pNK and term pregnancy dNK. Further- plays a role in pNK development, maturation, and function (41, more, mRNA for EOMES, GATA3, ITGA1, and CD56 were 42). TBX21 was shown to regulate IFN-g production in NK cells, increased in first trimester dNK. The term pregnancy dNK profile and IFN-g production was shown to be impaired in the absence of includes high expression of the dNK2 markers ITGB2 and T-bet (43). In this study, we show that first trimester dNK have the GZMH. The high NKG2R expression combined with low KIR lowest expression of TBX21 and IFN-g but the highest levels of expression in term dNK also reflect the dNK2 profile. However, EOMES, indicating EOMES does not increase IFN-g in dNK. in contrast to the dNK2 profile, nearly all term pregnancy dNK Furthermore, a positive correlation between TBX21 and PFN in express the cytolytic molecules PFN and GZMB. Further in- human NK cells was shown (44), and NK cells were shown to be vestigation is required on the topics of how these dNK subtypes less cytotoxic in the absence of TBX21; some have suggested this may change during pregnancy and which factors determine their is possibly caused by a decreased production of PFN and GZMB mechanism of local expansion or influx to decidual tissues. (42, 43). In this study first trimester dNK degranulation to K562 10 TERM PREGNANCY dNK ARE A DISTINCT TYPE OF NK CELLS and PMA/I was lower than pNK, but PFN and GZMB were pre- It is of high clinical relevance to determine whether there are sent in nearly 100% of dNK. GATA3 is another critical tran- dNK populations responsible for trophoblast invasion that are scriptional regulator of NK cell maturation and egress out of the distinct from subsets of dNK with effector functions ready to bone marrow (45). GATA3 deficiency did not result in reduced NK confront incoming pathogens. RNA sequencing on single cells or cell frequencies but led to reduced T-bet expression and IFN-g small dNK populations combined with functional assessment of production (46). Further investigations into the transcriptional and these dNK subsets can help identify functionally distinct dNK posttranscriptional regulatory mechanisms that contribute to spe- populations and determine their dynamics throughout pregnancy. A cialized dNK functions and subtypes throughout human pregnancy major challenge in this study is to connect gene expression analysis at is required to understand how dNK acquire unique characteristics the single-cell level with accurate assessment of dNK function. This to interact with trophoblasts and provide immunity. is particularly challenging for human dNK subtypes that have limited Numerous studies suggest that interactions through maternal survival potential in vitro and may proliferate in vivo but not in vitro. KIR expressed by dNK with paternal HLA-C expressed on EVT Further identification and validation of markers that can separate influence pregnancy success (16, 24). Whereas we demonstrate dNK types is required to determine whether and how dNK failure to that HLA-C is expressed by EVT from first trimester and term facilitate trophoblast invasion contributes to placental pathology and pregnancy as well as on HLA-G+ chorionic trophoblasts, the in- syndromes characterized by underdeveloped placentation, such as crease in KIR with specificity for HLA-C is restricted to the first preeclampsia and intrauterine growth restriction. Monitoring dNK trimester. This may suggest that the contribution of KIR/HLA-C activity to provide immunity to infection will contribute to identifying interactions specifically contribute to first trimester biological risk factors and protective factors for clearance of placental infection processes such as trophoblast invasion and spiral artery remodel- and transmission of pathogens from mothers to their unborn children. Downloaded from ing. This is in line with the previously found genetic correlations between the presence of maternal HLA-C–activating receptor Acknowledgments KIR2DS1 and HLA-C2 that reduces the risk for pregnancy We thank Joyce Lavecchio and Nema Kheradmand for help with cell complications, including preeclampsia. The finding that KIR2DS1 sorting; Donald Coen, Harvard Medical School, Boston, MA, for providing may increase the capacity of first trimester dNK to clear HCMV- HCMV-AD169-GFP; Ada Taymoori and the research team at Tufts Med-

infected DSC expressing HLA-C may contribute to the increased ical Center for all efforts collecting placental materials; and all past and http://www.jimmunol.org/ HCMV response by first trimester dNK compared with term current laboratory members for helpful discussions. pregnancy dNK (14). The binding of HCMV peptides in HLA-C2 molecules favored NK activation through KIR2DS1 over NK in- Disclosures hibition through KIR2DL1 (47). The authors have no financial conflicts of interest. In addition to dNK–EVT interactions through HLA-C, dNK also interact with HLA-G molecules expressed on EVT through KIR2DL4, LILRB1, and LILRB2 (HLA-G) (27). Interestingly, References 1. Koopman, L. A., H. D. Kopcow, B. Rybalov, J. E. Boyson, J. S. Orange, term pregnancy EVT had the highest expression levels of HLA-G, F. Schatz, R. Masch, C. J. Lockwood, A. D. Schachter, P. J. Park, and and the dNK in the d.basalis expressed KIR2DL4 (detected by J. L. Strominger. 2003. Human decidual natural killer cells are a unique by guest on September 29, 2021 QPCR and FACS) and LILRB2 (detected by FACS but not QPCR). NK cell subset with immunomodulatory potential. J. Exp. Med. 198: 1201–1212. First trimester EVT had lower expression levels of HLA-G, and 2. Kopcow, H. D., D. S. Allan, X. Chen, B. Rybalov, M. M. Andzelm, B. Ge, and first trimester dNK expressed all three HLA-G receptors, KIR2DL4, J. L. Strominger. 2005. Human decidual NK cells form immature activating LILRB1, and LILRB2, whereas dNK in d.parietalis expressed both synapses and are not cytotoxic. Proc. Natl. Acad. Sci. USA 102: 15563–15568. 3. King, A., T. Burrows, S. Verma, S. Hiby, and Y. W. Loke. 1998. Human KIR2DL4 and LILRB2. How this differential expression of HLA-G uterine lymphocytes. Hum. Reprod. Update 4: 480–485. and HLA-G receptors affects EVT and dNK interactions and their 4. Kopcow, H. D., M. Eriksson, T. F. Mselle, S. M. Damrauer, C. R. Wira, C. L. Sentman, and J. L. Strominger. 2010. Human decidual NK cells from subsequent function remains to be determined. Previous studies gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets. showed that KIR2DL4 and HLA-G interactions limit NK cytotox- Placenta 31: 334–338. icity but allow for IFN-g induction (48). These HLA-G receptors 5. Siewiera, J., H. El Costa, J. Tabiasco, A. Berrebi, G. Cartron, P. Le Bouteiller, and N. Jabrane-Ferrat. 2013. Human cytomegalovirus infection elicits new de- can also contribute to a phenomenon called trogocytosis, whereby cidual natural killer cell effector functions. [Published erratum appears in 2013 dNK directly acquire HLA-G from the EVT, incorporate HLA-G in PLoS Pathog. 9.] PLoS Pathog. 9: e1003257. the dNK membrane, and temporarily alter dNK function (15). 6. Hanna, J., D. Goldman-Wohl, Y. Hamani, I. Avraham, C. Greenfield, S. Natanson-Yaron, D. Prus, L. Cohen-Daniel, T. I. Arnon, I. Manaster, et al. Interactions of CD94/NKG2Rs with HLA-E molecules largely 2006. Decidual NK cells regulate key developmental processes at the human provides inhibition of NK cells. Cellular stress or intracellular fetal-maternal interface. Nat. Med. 12: 1065–1074. pathogens can prevent HLA-E cell surface expression and thus 7. Moffett-King, A. 2002. Natural killer cells and pregnancy. [Published erratum appears in 2002 Nat. Rev. Immunol. 2: 975.] Nat. Rev. Immunol. 2: 656–663. releases this inhibitory pathway and skews NK cells toward ac- 8. Erlebacher, A. 2013. Immunology of the maternal-fetal interface. Annu. Rev. tivation (25). Although CD94/NKG2A/CRs are increased on dNK Immunol. 31: 387–411. 9. Williams, P. J., R. F. Searle, S. C. Robson, B. A. Innes, and J. N. Bulmer. 2009. compared with pNK, no significant changes were observed be- Decidual leucocyte populations in early to late gestation normal human preg- tween first trimester and term pregnancy. Interestingly, NKG2D, a nancy. J. Reprod. Immunol. 82: 24–31. receptor for the cellular stress ligand MICA/B, was increased on 10. Sindram-Trujillo, A. P., S. A. Scherjon, P. P. van Hulst-van Miert, J. J. van Schip, H. H. H. Kanhai, D. L. Roelen, and F. H. J. Claas. 2003. Differential distribution term pregnancy dNK, giving them a possible greater ability to of NK cells in decidua basalis compared with decidua parietalis after uncom- detect and respond to subviable and stressed cells (49). Although plicated human term pregnancy. Hum. Immunol. 64: 921–929. this study presents comprehensive phenotypic, functional, and gene 11. Takahashi, H., T. Yamamoto, M. Yamazaki, T. Murase, T. Matsuno, and F. Chishima. 2018. Natural cytotoxicity receptors in decidua natural killer cells expression data to identify key differences between pNK, first tri- of term normal pregnancy. J. Pregnancy 2018: 4382084. mester dNK, and dNK at term pregnancy, further studies should in- 12. Sańchez-Rodrı´guez, E. N., S. Nava-Salazar, C. A. Mendoza-Rodrı´guez, C. Moran, J. F. Romero-Arauz, E. Ortega, J. Granados, A. Cervantes-Peredo, and vestigate how these differences relate to additional clinical variables, M. Cerboń. 2011. Persistence of decidual NK cells and KIR genotypes in healthy including but not limited to maternal–fetal HLA-C typing and HLA pregnant and preeclamptic women: a case-control study in the third trimester of mismatches, fetal sex, and the number of previous pregnancies. Par- gestation. Reprod. Biol. Endocrinol. 9: 8. 13. King, A., P. Wooding, L. Gardner, and Y. W. Loke. 1993. Expression of perforin, ticularly, HLA-C typing and the number of previous pregnancies have granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are been shown to influence dNK composition and function (50, 51). activated and capable of effector functions. Hum. Reprod. 8: 2061–2067. The Journal of Immunology 11

14. Crespo, A. C., J. L. Strominger, and T. Tilburgs. 2016. Expression of KIR2DS1 34. Salvany-Celades, M., A. van der Zwan, M. Benner, V. Setrajcic-Dragos, by decidual natural killer cells increases their ability to control placental HCMV H. A. Bougleux Gomes, V. Iyer, E. R. Norwitz, J. L. Strominger, and T. Tilburgs. infection. Proc. Natl. Acad. Sci. USA 113: 15072–15077. 2019. Three types of functional regulatory T cells control T cell responses at the 15. Tilburgs, T., J. H. Evans, A. C. Crespo, and J. L. Strominger. 2015. The HLA-G human maternal-fetal interface. Cell Rep. 27: 2537–2547.e5. cycle provides for both NK tolerance and immunity at the maternal-fetal inter- 35. van der Zwan, A., K. Bi, E. R. Norwitz, A. C. Crespo, F. H. J. Claas, face. Proc. Natl. Acad. Sci. USA 112: 13312–13317. J. L. Strominger, and T. Tilburgs. 2018. Mixed signature of activation and 16. Xiong, S., A. M. Sharkey, P. R. Kennedy, L. Gardner, L. E. Farrell, dysfunction allows human decidual CD8+ T cells to provide both tolerance and O. Chazara, J. Bauer, S. E. Hiby, F. Colucci, and A. Moffett. 2013. Maternal immunity. Proc. Natl. Acad. Sci. USA 115: 385–390. uterine NK cell-activating receptor KIR2DS1 enhances placentation. J. Clin. 36. Blanco, O., I. Tirado, R. Munõz-Fernańdez, A. C. Abadıá-Molina,J.M.Garcıá- Invest. 123: 4264–4272. Pacheco, J. Penã, and E. G. Olivares. 2008. Human decidual stromal cells express 17. Tilburgs, T., A. C. Crespo, A. van der Zwan, B. Rybalov, T. Raj, B. Stranger, HLA-G: effects of cytokines and decidualization. Hum. Reprod. 23: 144–152. L. Gardner, A. Moffett, and J. L. Strominger. 2015. Human HLA-G+ extravillous 37. Tilburgs, T., D. Schonkeren, M. Eikmans, N. M. Nagtzaam, G. Datema, trophoblasts: immune-activating cells that interact with decidual leukocytes. G. M. Swings, F. Prins, J. M. van Lith, B. J. van der Mast, D. L. Roelen, et al. Proc. Natl. Acad. Sci. USA 112: 7219–7224. 2010. Human decidual tissue contains differentiated CD8+ effector-memory 18. Vujaklija, D. V., T. Gulic, S. Sucic, K. Nagata, K. Ogawa, G. Laskarin, S. Saito, T cells with unique properties. J. Immunol. 185: 4470–4477. H. Haller, and D. Rukavina. 2011. First trimester pregnancy decidual natural 38. Montaldo, E., P. Vacca, L. Chiossone, D. Croxatto, F. Loiacono, S. Martini, killer cells contain and spontaneously release high quantities of granulysin. Am. S. Ferrero, T. Walzer, L. Moretta, and M. C. Mingari. 2016. Unique Eomes(+) J. Reprod. Immunol. 66: 363–372. NK cell subsets are present in uterus and decidua during early pregnancy. Front. 19. Barman, H., M. Walch, S. Latinovic-Golic, C. Dumrese, M. Dolder, Immunol. 6: 646. P. Groscurth, and U. Ziegler. 2006. Cholesterol in negatively charged lipid bi- 39. Vacca, P., E. Montaldo, C. Vitale, D. Croxatto, L. Moretta, and M. C. Mingari. layers modulates the effect of the antimicrobial protein granulysin. J. Membr. 2015. MSC and innate immune cell interactions: a lesson from human decidua. Biol. 212: 29–39. 20. Papućhova´, H., T. B. Meissner, Q. Li, J. L. Strominger, and T. Tilburgs. 2019. Immunol. Lett. 168: 170–174. The dual role of HLA-C in tolerance and immunity at the maternal-fetal inter- 40. Allan, D. S., B. Rybalov, G. Awong, J. C. Zuń˜iga-Pflu¨cker, H. D. Kopcow, face. Front. Immunol. DOI: 10.3389/fimmu.2019.02730. J. R. Carlyle, and J. L. Strominger. 2010. TGF-b affects development and 21. Hiby,S.E.,R.Apps,A.M.Sharkey,L.E.Farrell,L.Gardner,A.Mulder,F.H.Claas, differentiation of human natural killer cell subsets. Eur. J. Immunol. 40: Downloaded from J. J. Walker, C. W. Redman, L. Morgan, et al. 2010. Maternal activating KIRs protect 2289–2295. against human reproductive failure mediated by fetal HLA-C2. [Published erratum 41. Simonetta, F., A. Pradier, and E. Roosnek. 2016. T-bet and eomesodermin in NK appears in 2011 J. Clin. Invest. 121: 455.] J. Clin. Invest. 120: 4102–4110. cell development, maturation, and function. Front. Immunol. 7: 241. 22. Sharkey, A. M., L. Gardner, S. Hiby, L. Farrell, R. Apps, L. Masters, 42. Gordon, S. M., J. Chaix, L. J. Rupp, J. Wu, S. Madera, J. C. Sun, T. Lindsten, and J. Goodridge, L. Lathbury, C. A. Stewart, S. Verma, and A. Moffett. 2008. Killer S. L. Reiner. 2012. The transcription factors T-bet and Eomes control key Ig-like receptor expression in uterine NK cells is biased toward recognition of checkpoints of natural killer cell maturation. Immunity 36: 55–67. HLA-C and alters with gestational age. J. Immunol. 181: 39–46. 43. Townsend, M. J., A. S. Weinmann, J. L. Matsuda, R. Salomon, P. J. Farnham,

23. Foley, B., D. De Santis, L. Lathbury, F. Christiansen, and C. Witt. 2008. C. A. Biron, L. Gapin, and L. H. Glimcher. 2004. T-bet regulates the terminal http://www.jimmunol.org/ KIR2DS1-mediated activation overrides NKG2A-mediated inhibition in HLA-C maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 20: C2-negative individuals. Int. Immunol. 20: 555–563. 477–494. 24. Hiby, S. E., J. J. Walker, K. M. O’shaughnessy, C. W. Redman, M. Carrington, 44. Simonetta, F., A. Pradier, C. Bosshard, S. Masouridi-Levrat, Y. Chalandon, and J. Trowsdale, and A. Moffett. 2004. Combinations of maternal KIR and fetal E. Roosnek. 2015. NK cell functional impairment after allogeneic hematopoietic HLA-C genes influence the risk of preeclampsia and reproductive success. stem cell transplantation is associated with reduced levels of T-bet and eome- J. Exp. Med. 200: 957–965. sodermin. J. Immunol. 195: 4712–4720. 25. Long, E. O., H. S. Kim, D. Liu, M. E. Peterson, and S. Rajagopalan. 2013. 45. Ali, A. K., J. S. Oh, E. Vivier, M. Busslinger, and S. H. Lee. 2016. NK cell- Controlling natural killer cell responses: integration of signals for activation and specific Gata3 ablation identifies the maturation program required for bone inhibition. Annu. Rev. Immunol. 31: 227–258. marrow exit and control of proliferation. J. Immunol. 196: 1753–1767. 26. Rajagopalan, S., Y. T. Bryceson, S. P. Kuppusamy, D. E. Geraghty, A. van der 46. Samson, S. I., O. Richard, M. Tavian, T. Ranson, C. A. Vosshenrich, F. Colucci, Meer, I. Joosten, and E. O. Long. 2006. Activation of NK cells by an endocy- J. Buer, F. Grosveld, I. Godin, and J. P. Di Santo. 2003. GATA-3 promotes

tosed receptor for soluble HLA-G. PLoS Biol. 4: e9. maturation, IFN-gamma production, and liver-speciﬁc homing of NK cells. by guest on September 29, 2021 27. Li, C., B. L. Houser, M. L. Nicotra, and J. L. Strominger. 2009. HLA-G Immunity 19: 701–711. homodimer-induced cytokine secretion through HLA-G receptors on human 47. van der Ploeg, K., C. Chang, M. A. Ivarsson, A. Moffett, M. R. Wills, and decidual macrophages and natural killer cells. Proc. Natl. Acad. Sci. USA 106: J. Trowsdale. 2017. Modulation of human leukocyte antigen-C by human cy- 5767–5772. tomegalovirus stimulates KIR2DS1 recognition by natural killer cells. Front. 28. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16: 359–393. Immunol. 8: 298. 29. Biassoni, R. 2008. Natural killer cell receptors. Adv. Exp. Med. Biol. 640: 35–52. 48. Rajagopalan, S., J. Fu, and E. O. Long. 2001. Cutting edge: induction of IFN- 30. Horowitz, A., D. M. Strauss-Albee, M. Leipold, J. Kubo, N. Nemat-Gorgani, O. C. Dogan, C. L. Dekker, S. Mackey, H. Maecker, G. E. Swan, et al. 2013. gamma production but not cytotoxicity by the killer cell Ig-like receptor Genetic and environmental determinants of human NK cell diversity revealed by KIR2DL4 (CD158d) in resting NK cells. J. Immunol. 167: 1877–1881. mass cytometry. Sci. Transl. Med. 5: 208ra145. 49. Champsaur, M., and L. L. Lanier. 2010. Effect of NKG2D ligand expression on 31. Vento-Tormo, R., M. Efremova, R. A. Botting, M. Y. Turco, M. Vento-Tormo, host immune responses. Immunol. Rev. 235: 267–285. K. B. Meyer, J. E. Park, E. Stephenson, K. Polanski, A. Goncalves, et al. 2018. 50. Tilburgs, T., S. A. Scherjon, B. J. van der Mast, G. W. Haasnoot, M. Versteeg-V Single-cell reconstruction of the early maternal-fetal interface in humans. Nature D Voort-Maarschalk, D. L. Roelen, J. J. van Rood, and F. H. Claas. 2009. Fetal- 563: 347–353. maternal HLA-C mismatch is associated with decidual T cell activation and 32. Sojka, D. K., L. Yang, B. Plougastel-Douglas, D. A. Higuchi, B. A. Croy, and induction of functional T regulatory cells. J. Reprod. Immunol. 82: 148–157. W. M. Yokoyama. 2018. Cutting edge: local proliferation of uterine tissue- 51. Gamliel, M., D. Goldman-Wohl, B. Isaacson, C. Gur, N. Stein, R. Yamin, resident NK cells during decidualization in mice. J. Immunol. 201: 2551–2556. M. Berger, M. Grunewald, E. Keshet, Y. Rais, et al. 2018. Trained memory of 33. Sojka, D. K., L. Yang, and W. M. Yokoyama. 2019. Uterine natural killer cells. human uterine NK cells enhances their function in subsequent pregnancies. Front. Immunol. 10: 960. Immunity 48: 951–962.e5. KIR2DS1- KIR2DS1+ A blood

decidua 6-12wk blood IgG IgG blood 6-12wk

decidua d.basalis >37wk >37wk S1 d.basalis d.parietalis >37wk KIR2D >37wk d.parietalis d.parietalis KIR2DL1 KIR2DL2/3 KIR2DL1

B 6-12wk decidua >37wk blood IgG IgG blood >37wk d.basalis

blood

>37wk decidua 6-12wk

d.parietalis d.parietalis CD94 NKG2A NKG2C NKG2D d.basalis >37wk C d.parietalis >37wk

Supplementary Figure S1. KIR2D expression is increased in 1st trimester dNK but not in term pregnancy dNK. A) Representative FACS histograms of KIR2DL1 andKIR2DL2/3 and dotplots of KIR2DL1 and KIR2DL1/KIR2DS1 expression on pNK and dNK from 6-12wk decidua, decidua basalis >37wk and decidua parietalis >37wk. B) Representative FACS histograms and C) Graphs of CD94, NKG2A, NKG2C and NKG2D expression compared to IgG control on freshly isolated pNK and dNK from 6-12wk decidua, decidua basalis >37wk and decidua parietalis >37wk. N = 5-8; Bars represent median and interquartile range; *P<0.05, **P<0.01 and ***P<0.001. A lo IgG blood 6-12wk decidua >37wk d.basalis >37wk

d.parietalis d.parietalis NKp30 NKp46 CD57 2B4 DNAM1

B 100 20 100 25

80 80 20 *** 15 * 60 60 15 10 40 40 10

5 20 20 5

0 0 0 0 C 100 20 20 * *** blood 80 ** * ** ** 15 *** 15 decidua 6-12wk * d.basalis >37wk 60 d.parietalis >37wk 10 10 40

5 5 20

0 0 0

Supplementary Figure S2. Differential NK receptor expression by pNK and dNK. A) Representative FACS histograms of Nkp30, NKp46, CD57, 2B4 and DNAM1 expression on freshly isolated pNK and dNK from 6-12wk decidua, decidua basalis >37wk and decidua parietalis >37wk. B) Graphs depict percentage and MFI of Nkp30 and NKp46 and percentage of CD57, 2B4 and DNAM1 expression on pNK and dNK. N= 6-8; Bars represent median and interquartile range. *P<0.05, **P<0.01 and ***P<0.001. A

D blood decidua 6-12wk d.basalis >37wk d.parietalis >37wk

Supplementary Figure S3. Gene expression profiles of pNK and dNK. Relative mRNA expression for genes included in A) Fig 6 cluster I, B) Fig 6 cluster II, C) Fig 6 cluster III, D) Fig 6 cluster IV. Bars represent mean and SEM for N = 8 for all NK cell types; *P<0.05, **P<0.01 and ***P<0.001. Relative Expression: pNK dNK Low High 6-12wk 37wk decidua d.basalis d.parietalis Cytolytic granules %PFN+ cells MFI PFN %GZMB+ cells MFI GZMB %GNLY+ cells MFI GNLY %9kDa GNLY+ cells MFI 9kDa GNLY GZMK (mRNA) GZMH (mRNA) Degranulation PMA CD107a K562 HCMV NK receptors HLA-C KIR2DL1 KIR2DL2/3 KIR2DS1 HLA-E CD94 NKG2A NKG2C HLA-G KIR2DL4 (IC) KIR2DL4 (mRNA) LILRB1 LILRB1 (mRNA) LILRB2 LILRB2 (mRNA) MICA/B NKG2D Other %NKp30+ cells MFI NKp30 %NKp46+ cells MFI NKp46 NKp44 (mRNA) NKp80 (mRNA) KLRG1 (mRNA) KIR3DL1 mRNA) CD56 (mRNA) CD57 DNAM 2B4 Cytokines IFNɣ IFNɣ (mRNA) TNFɑ TNFɑ (mRNA) CSF2 (mRNA) VEGFA (mRNA) Transcription factors TBX21 (mRNA) EOMES (mRNA) GATA3 (mRNA) Other PDCD1 (mRNA) CD69 (mRNA) IFNGR1 (mRNA) ITGA1 (mRNA) ITGB2 (mRNA)

Supplementary Table S1. Phenotypic and functional features of pNK and dNK.