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. 2011 Sep 7;86(1):9. doi: 10.1095/biolreprod.111.094383

Immunophenotype and Cytokine Profiles of Rhesus Monkey CD56bright and CD56dim Decidual Natural Killer Cells1

Svetlana V Dambaeva 3, Maureen Durning 4, Ann E Rozner 3, Thaddeus G Golos 3,4,5,2
PMCID: PMC3313663  PMID: 21900681

ABSTRACT

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56bright natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56bright CD16+NKp30− decidual NK cells, with minor CD56dim and CD56neg dNK cells. Intracellular cytokine staining demonstrated that CD56dim and not CD56bright dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.

Keywords: decidua, endometrium, interferon-gamma (IFNG), natural killer cell, pregnancy, primates, reproductive immunology, rhesus monkey

Decidual CD56bright and CD56dim NK cells in the rhesus monkey differ in IFNG and IL6 expression and are maintained, along with other leukocyte subsets, in characteristic proportions within individual animals across successive pregnancies.

INTRODUCTION

With the onset of pregnancy, the human and nonhuman primate endometrium undergoes a series of remarkable changes, including a substantial increase in the number of immunocompetent cells populating this uterine mucosal lining. It is well established that natural killer (NK) cells are the dominant immune cell type detected in the human and monkey decidua (endometrium of pregnancy) and constitute up to 70% of all leukocytes [1–3]. One of the most important similarities of human and nonhuman primate placentation is the invasion by trophoblasts of the uterine stroma and vessels. The abundant decidual NK (dNK) cells are relevant candidates for interaction with trophoblasts to achieve this invasion. Decidual NK cells express killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs), and CD94/NKG2, receptors that are known to recognize a restricted repertoire of human trophoblast MHC class I molecules (HLA-C, -G, and -E) [4]. In addition to protecting trophoblasts from NK cell cytotoxic activity, the interaction with HLA-G and HLA-E could influence cytokine production by dNK cells [5–7]. Studies assaying NK cell secretory products in culture media or by intracellular cytokine staining revealed that dNK cells produce a variety of cytokines, chemokines, and growth factors, including angiogenic growth factors: interleukin 6 (IL6), transforming growth factor B1, vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), angiopoietin (ANG) 1, ANG2, C-X-C ligand (CXCL) 8 (IL8), and CXCL10 (IP10) [7–10]. Thus, it is considered that dNK cells play an important role at the maternal-fetal interface, participating in the regulation of trophoblast invasion and endometrial angiogenesis.

There are two subsets of human peripheral blood NK (pNK) cells: the major population of pNK cells (∼95%) are cytotoxic CD56dimCD16+ cells, whereas ∼5% of pNK cells are CD56brightCD16− cells, which are characterized by low cytotoxic activity but greater cytokine production, and have been referred to as immunoregulatory cells [11]. Studies were performed to reveal that dNK cells are a distinct NK cell subset [12]. They have been generally considered as a homogeneous population, which display a phenotype similar to the CD56bright pNK cells in terms of high CD56 expression and negativity for CD16, but resemble CD56dim pNK cells in their KIR expression level and granularity. In addition, dNK cells are characterized by differential expression of a large set of genes, including genes encoding tetraspanins, integrin subunits, and galectins [12]. However, there are also studies where a CD56dimCD16+ population of dNK cells has been noted, including in association with pregnancy failure [10, 13, 14], but it is not clear if this population is different from pNK cells.

In the present study we analyzed rhesus decidual leukocyte populations in early pregnancy to demonstrate the phenotype and functional differences among the different dNK cell subsets. Cytokine secretion and proliferation marker expression revealed the dynamic nature of these cells during the establishment of pregnancy. In addition, we found that the relative proportion of the subsets of dNK cells and overall leukocyte populations was characteristic of individual animals and consistent across multiple pregnancies, perhaps suggesting a genetic component that carefully controls the decidual immune environment.

MATERIALS AND METHODS

Animals

Female rhesus macaques (Macaca mulatta) used for timed mating were from the colony maintained at the Wisconsin National Primate Research Center. All procedures with animals were performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, and under approval of the University of Wisconsin-Madison Graduate School Animal Care and Use Committee.

Isolation of Peripheral Blood and Decidual Mononuclear Leukocytes

Venous peripheral blood was drawn into vacutainers containing Na-heparin (BD, Franklin Lakes, NJ). Blood was diluted 1:2 with RPMI-1640 and layered on top of Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) followed by density gradient centrifugation for mononuclear leukocyte (MNL) separation. Decidual tissues were obtained by fetectomy at Days 35–38, with Day 0 the day after the luteinizing hormone surge. This stage of pregnancy is approximately equivalent to 8–9 wk of human pregnancy, when dNK cells are closely associated with maternal spiral arterioles in the decidua, and are thought to contribute to their active remodeling [1, 15]. A total of 23 decidual samples were used in the analysis from 14 animals, with eight of the animals providing tissues from more than one fetectomy (see Supplemental Table S1 for information on individual animals; all Supplemental Data are available online at www.biolreprod.org). The decidua was dissected free of the amniotic membranes and the placenta, weighed, minced, and digested with a collagenase IV/DNAse cocktail as previously described [2]. MNLs from the suspension of decidual cells were separated on a gradient of Ficoll-Paque Plus, and viable cell number was determined using trypan blue dye exclusion.

Flow Cytometry

Up to eight-color flow cytometry analysis was performed. The antibodies and staining panels used in the study are listed in Table 1 and Supplemental Table S2, respectively. A combination of monoclonal antibodies (mAbs) against CD45, CD56, CD3, CD14, and CD16 was included in each sample to gate decidual immune cell populations. Other mAbs were added to the gating combination for further phenotyping or intracellular cytokine analysis. For the isotype controls, specific mAbs were substituted for corresponding nonspecific IgG isotypes. In addition, control samples containing the gating combination of mAbs along with IgG isotypes for the rest of mAbs were prepared. Eight-color panels included LIVE/DEAD Fixable Violet stain (ViVID; Invitrogen, Eugene, OR) to discriminate dead cells. Labeling was performed in 96-well U-bottom plates. MNLs at 2.5–3 × 105 cells in 50 μl of PBS with 2% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) or plain PBS (if ViVID was employed) were added to a cocktail of mAbs against surface markers and ViVID. After 25 min incubation at 4°C, cells were washed twice, then fixed/permeabilized with Cytofix/Cytoperm, 100 μl/well, and washed twice with Perm/Wash buffer (both from BD Pharmingen, San Diego, CA). Samples that required intracellular staining were next stained with relevant mAbs. MAbs against cytokines, perforin, and MKI67 were applied at this step. After 25 min incubation at 4°C, cells were washed twice with Perm/Wash buffer. All samples were subsequently transferred into 5-ml Falcon tubes (BD, Bedford, MA) in 300 μl Perm/Wash buffer and processed on LSR-II (BD, San Jose, CA) with collection of 30 000 events in the leukocyte gate. Compensation controls were prepared using AbC anti-mouse bead kit for mouse mAb capture and ArC amine reactive compensation bead kit for ViVID (both kits from Invitrogen), according to the manufacturer's instructions. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR), where compensation matrices based on compensation controls were computed for each separate mAb combination and applied to the appropriate samples. To estimate the absolute number of leukocytes per gram decidua the following formula was used: (total mononuclear cell count/tissue weight) × (% of CD45+ cells/100).

TABLE 1.

mAbs used in flow cytometry analysis of rhesus monkey decidual and peripheral blood cells.

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In Vitro Stimulation for CD16 Expression and Intracellular Cytokine Analysis

Freshly isolated decidual MNLs were resuspended at 2 × 106 cells/ml in complete medium, which is RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml)-streptomycin (100 μg/ml), and 55 μM 2-mercaptoethanol (Gibco, Grand Island, NY). Cells were incubated for 5 h in the presence of 50 ng/ml phorbol myristyl acetate (PMA), 1 μg/ml ionomycin, and 0.1 μg/ml lipopolysaccharide (LPS) (all from Sigma, St. Louis, MO), or without stimulation agents in control samples. To analyze intracellular accumulation of cytokines, Brefeldin A, 10 μg/ml (Sigma), was added for the last 4 h. After the incubation, a cell scraper was applied to ensure collection of all cells including macrophages; cells then were washed with PBS and processed for flow cytometry staining as described above.

Isolation of dNK Subsets and pNK Cells

Subsets of dNK cells were sorted from the freshly isolated decidual leukocyte suspension using immunomagnetic separation. Decidual MNLs were incubated with CD20 fluorescein isothiocyanate (FITC) mAb for 25 min at 4°C, washed twice in cold PBS with 2% FBS, and magnetically labeled with anti-FITC microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. CD20+ cells, which represent in decidua CD56dim dNK cells (see Results), were positively selected using an LS column and MidiMACS separation unit (Miltenyi Biotec). CD56bright dNK cells were sorted from the CD20-negative fraction with nonhuman primate anti-CD56 microbeads (Miltenyi Biotec). The pNK cells were sorted from peripheral blood MNLs using nonhuman primate anti-CD16 microbeads (Miltenyi Biotec). A sample of every sorted fraction was set aside for flow cytometry analysis. The average purity of sorting was over 73% for CD56dim dNK cells, over 76% for the CD56bright dNK cell fraction, and over 95% for pNK cells. The CD20−CD56− decidual fraction was mostly depleted of NK cells, but enriched with T cells (38.6% ± 12.7%) and macrophages (24.2% ± 8.9%).

Medium Analysis for Cytokine Secretion by dNK Cells Subsets and pNK Cells

NK cells or NK cell-depleted decidual leukocytes were seeded into 96-well plates for 24 h at 50 × 103 cells/well in 200 μl of complete medium. Control wells had only medium. CD56bright, CD56dim dNK cells, or the NK cell-depleted fraction of decidual leukocytes was cultured in the presence or absence of 5 ng/ml of IL15, and pNK cells in the presence or absence of 500 U/ml of IL2 (both cytokines from Peprotech, Rocky Hill, NJ). Supernatants from cultures were collected and stored at −80°C until cytokines were measured using a Milliplex nonhuman primate cytokine kit (Millipore, Billerica, MA) according to the manufacturer's instructions.

Statistical Analysis

Statistical analysis was done using GraphPad Prism software (GraphPad Software, La Jolla, CA). Mann-Whitney or Wilcoxon (nonparametric, paired) tests of cytokine secretion and NKp46 expression, or correlation analyses of data in Table 2 with the Spearman rank correlation method with a two-tailed distribution, were used where indicated. Significant differences between the percentages of CD45+, CD56bright, or CD56dim NK cells in repeated fetectomies within a given animal was tested by Wilcoxon signed rank test. To compare between- and within-subject variability in the percentage of decidual CD56bright and CD56dim cells, a one-way random-effect ANOVA model was used. P values < 0.05 indicated significant differences.

TABLE 2.

Correlation coefficients between different data sets.*

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RESULTS

Three Subsets of dNK Cells Are Detected in Early Pregnancy Rhesus Decidua: CD56bright, CD56dim, and CD56neg dNK Cells

Multicolor flow cytometry analysis of freshly isolated decidual cells from Day 35–38 gestation tissue allowed simultaneous identification of NK cells, T cells, and macrophages in a single sample. The gating strategy for immune cell detection is presented in Figure 1A. The majority of decidual CD45+ cells had high light-scatter characteristics and were CD56bright dNK cells and CD14+ macrophages (R1 population). The analysis of cells with a low light-scatter small lymphocyte profile (R2 population) revealed, in addition to CD3+ T cells, the presence of a CD3−CD56dim population. These cells were CD14 negative (not shown), which excludes the possibility of contamination by peripheral blood monocytes, which are CD56+ in macaques, and these cells were thus designated as CD56dim dNK cells. Within the CD3−CD56− population, an additional subtype of NK cells was identified by CD16 expression; these cells were designated as CD56neg dNK cells. CD56dim and CD56neg dNK cells comprise 16.7% ± 12.8% and 3.7% ± 2.8%, respectively, of the total population of dNK cells and could be considered as minor dNK cell subtypes. The general proportion of all immune cell populations in early pregnancy decidua, including CD14+ macrophages and CD3+ T cells, is shown in Figure 1B. Interestingly, animals with a higher proportion of the CD56dim subtype and lower proportion of the CD56bright subtype within dNK cells correlated with an increased percentage of T cells in the decidual leukocyte suspension (Fig. 1C and Table 2). The total percentage of dNK cells was not related to the number of T cells, but was associated with an increased number of macrophages instead (Table 2). On the other hand, no significant correlation with animal age, total numbers of pregnancies, or absolute number of leukocytes isolated from decidua was revealed on dNK cells, dNK cell subtypes, T cells, and macrophages (Table 2).

FIG. 1.

FIG. 1.

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Three dNK cell populations are distinguished in early pregnancy rhesus decidua. A) Gating strategy for decidual immune cell analysis by flow cytometry. We employed mAbs against CD45, CD56, CD14, CD3, and CD16 to define dNK cell populations. Within CD45+ cells two distinct fractions (R1 and R2) could be identified according to their light-scattering characteristics. CD56bright dNK cells were detected in R1 as CD14−CD56+ cells along with CD14+56− macrophages. CD56dim and CD56neg dNK cells were gated within R2 as CD3−56+ and CD16+56− cells, respectively. T cells were defined as CD3+ cells in R2. B) The percentage of dNK cells, T cells, and macrophages among CD45+ cells isolated from rhesus decidua at Days 36–38 of gestation. The results are the mean ± SD of 22 experiments. C) Correlation between the frequency of CD56dim dNK cells and the percentage of T cells in the decidua. Spearman rank correlation method with a two-tailed distribution was used.

The Decidual Immune Cell Profile Is Dam Specific and Consistent Across Pregnancies

There was variation between individuals in the ratio of dNK cell subtypes. Analysis in eight animals showed that animal-specific characteristics of dNK cell proportions were reproducible across two or three different pregnancies: if an animal had a high proportion of CD56bright or CD56dim dNK cells in a first pregnancy, values were not significantly different (P = 0.3) in her next pregnancy (e.g., animal r97095 vs. r01014; Fig. 2A). The between-animal variation was significantly greater than the within-animal variation for CD56dim NK cells (P < 0.01), and approached significance for CD56bright NK cells (P < 0.08). Moreover, it was discovered that the proportions of macrophages, T cells, and dNK cells were very close to those determined in the previous pregnancy in these animals (Fig. 2B). For example, the relatively high number of macrophages in the first pregnancy of animal r05014 was repeated in the next pregnancy. The absolute number of CD45+ cells isolated per gram decidua was estimated for 17 samples and found to be variable between animals (Fig. 2C). Strikingly, within individual animals the absolute numbers of decidual leukocytes across two or three pregnancies were not statistically different (P = 0.87) (Fig. 2C). Conversely, these percentages were more variable between animals than within animals (P < 0.01).

FIG. 2.

FIG. 2.

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Leukocyte analysis from repeated pregnancies in individual rhesus monkeys. A) The proportion of CD56bright, CD56dim, and CD56neg cells in the total dNK cell population from individual pregnancies. Labels on the x-axes are animals' ID and day of gestation at the time of tissue collection. For animal r04083, both pregnancies were with the same sire, whereas all other animals had different sires for each pregnancy. B) The composition of the decidual leukocyte population in individual animals. The percentages of macrophages, T cells, and dNK cells in total CD45+ decidual cells are presented. Mph, macrophage. Data are presented as in A. C) Absolute number of CD45+ cells isolated per gram of decidua across different animals. Fx, fetectomy. Labels on the x-axes are animals' identification numbers.

The Phenotypes of CD56bright and CD56dim dNK Cells Are Distinct

Beyond the light-scatter characteristic differences, all three dNK subsets revealed unique immunophenotypes, distinct from pNK cells (Fig. 3). In the rhesus monkey, pNK cells have a CD3-CD56-CD8+CD94/NKG2+NKp46+NKp30+ phenotype. The majority of rhesus pNK cells were CD16+. CD16 was not detectable on 6.8% ± 1.4% of pNK cells. Dim CD20 expression was discovered on 13.4% ± 5.8% (n = 10) of rhesus pNK cells regardless of CD16 expression. Analysis of a range of surface markers on dNK cells revealed that only NKG2/CD94 has consistent expression on all dNK cell subtypes. A human anti-CD159a (CD94/NKG2A) mAb (clone Z199), which cross-reacts with both rhesus NKG2A and NKG2C in combination with CD94 [16], was used in our study. In absolute contrast to pNK cells, a vast majority of dNK cells displayed bright or dim CD56 expression. CD56bright dNK cells were distinguished by no expression of NKp30 and a lower level of CD8 expression than on CD56dim dNK cells: 61.4% ± 31.9% vs. 82.8% ± 16.4% respectively (n = 17, P < 0.05). CD56dim dNK cells in turn were distinguished by the lower CD16 expression (19.2% ± 8.7% vs. 76.2% ± 15.6% in CD56bright dNK cells, n = 17, P < 0.001). Although only an extremely low number of CD20high B cells was found in the decidual MNL suspension, CD56dim dNK cells demonstrated CD20dim staining in the decidua (Fig. 3). Moreover, in contrast to other dNK cells and pNK cells, CD56dim dNK cells were characterized by the lowest perforin expression (Fig. 3). Finally, whereas few pNK cells or CD56neg dNK cells had detectable MKI67 expression, a majority of CD56dim and especially CD56bright dNK cells expressed this proliferation marker. The scarcest dNK cell subtype, CD56neg dNK cells, to some extent resembled pNK cells, but overall they had lower expression of CD8 and NCRs, suggesting that they are not identical to pNK cells. Subsequently, because of the very scant size of the CD56neg dNK cell population, our main focus in this study was on CD56bright and CD56dim dNK cells.

FIG. 3.

FIG. 3.

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Phenotypic characterization of dNK cell, pNK cell, and decidual T cell populations. The expression of the indicated markers was analyzed by six- or eight-color flow cytometry. CD56bright, CD56dim, and CD56neg dNK cells and decidual T cells (dec T cells) were gated as indicated in Figure 1A. pNK cells were identified within the lymphocyte gate as CD3−CD8+ or CD3−CD159a+ cells (not shown). The open black histograms represent staining with the indicated mAbs. The filled gray histograms represent isotype control staining. One representative is shown of 6–20 experiments with similar results.

CD56dim dNK Cells Revealed an Activated Phenotype

CD56bright and CD56dim dNK cells were found to be different when the expression of an activation marker, CD69, was assessed on freshly isolated decidual MNLs. Positive CD69 staining was revealed on CD56dim dNK cells, on T cells, and partially on CD56neg dNK cells, but CD56bright dNK cells were CD69 negative (Fig. 3). The lack of CD16 expression on CD56dim dNK cells may also indicate an activated status of these cells. We discovered that in vitro stimulation triggers the loss of CD16 from dNK cells (Fig. 4A). Decidual MNLs were incubated for 5 h with or without PMA, ionomycin, and LPS, as indicated in Materials and Methods. In contrast to the control sample, the stimulated sample had CD56bright (upper row) and CD56dim dNK cells (lower row) transformed into CD16-negative cells; in addition, CD56neg dNK cells (lower row) were not detectable. Another characteristic of CD56dim dNK cells as CD20dim-positive cells also can be linked with activated/armed status. The analysis of rhesus pNK cells revealed that CD20dim expression is associated with a significantly higher level of NKp46 expression (Fig. 4B). On the other hand, we did not detect a difference in the levels of CD16, CD69, CD159a, and NKp30 expression between CD20dim and CD20− pNK cells (data not shown).

FIG. 4.

FIG. 4.

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Low CD16 expression and dim CD20 expression on NK cells could be associated with activated phenotype. A) Loss of CD16 expression on decidual NK cells after stimulation. Decidual MNL suspension was incubated for the indicated time periods with or without stimulation (PMA + ionomycin + LPS). In upper row, R1 gated cells are shown; in lower row, R2 gated cells are shown; R1 and R2 were set as indicated in Figure 1A. One representative of four experiments is shown. B) Flow cytometry analysis of rhesus monkey whole blood stained with mAbs against NKp46 and CD20. Lymphocyte gate is shown. CD20− and CD20dim pNK cells are gated. A representative dot plot is shown of six animals tested. Column chart shows the level of NKp46 expression on CD20− and CD20dim pNK cell subpopulations. Presented are averages for mean intensity of fluorescence (MIF) ± SD of six animals tested, **P < 0.01 (Mann-Whitney U-test, nonparametric).

Single-Cell-Specific Analysis of Cytokine Expression: CD56dim dNK Cells but Not CD56bright Cells Are Interferon-Gamma Producers

To learn more about dNK activation status, cytokine production by decidual immune cells was analyzed by intracellular cytokine staining and flow cytometry (Fig. 5). Intracellular staining of stimulated decidual MNLs revealed interferon-gamma (IFNG)-expressing cells within CD56dim dNK cell and T cell populations, whereas CD56bright dNK cells and macrophages remained IFNG negative. Intracellular staining with anti-tumor necrosis factor (TNF) mAb showed that upon stimulation, T cells and macrophages were the main producers of this cytokine, whereas 78%–84% of dNK cells were negative.

FIG. 5.

FIG. 5.

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Single-cell analysis of cytokine expression. The decidual MNL suspension was incubated with PMA, ionomycin, and LPS for 5 h; Brefeldin A was added for the last 4 h. The expression of the indicated cytokines was analyzed by six- or eight-color flow cytometry. CD56bright and CD56dim dNK cells, T cells, and macrophages were gated as indicated in Figure 1A. Black open histograms represent expression for indicated cytokines. Gray filled histograms represent isotype control staining. IgG isotypes were used along with all surface markers to gate the relevant populations for control expression. One representative is shown of six experiments.

Cytokine Profile of CD56dim and CD56bright dNK Cells Is Distinct from pNK Cells

The clear differential production of IFNG and TNF prompted further analysis of the secretion of cytokines and growth factors into medium by separate dNK cells subtypes. CD56dim dNK cells were selected from the decidual MNL suspension owing to their unique CD20 expression using magnetic immunolabeling (Fig. 6). Next, anti-CD56 microbeads were applied to select remaining CD56-positive cells. The resulting enriched CD56dim and CD56bright dNK cell fractions were cultured for 24 h and supernatants were collected for cytokine analysis by Luminex assay. In addition, supernatants from pNK cells and from CD20−CD56− (NK cell-depleted fraction) decidual leukocytes were prepared and analyzed the same way.

FIG. 6.

FIG. 6.

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Separation of CD56dim and CD56bright dNK cells. Decidual cells were first labeled with CD20-FITC mAb and subsequently magnetically labeled with anti-FITC microbeads and separated on a MACS column. The CD20+ cells are an enriched CD56dim dNK cell population (marked as tinted gate in CD20+ fraction). CD56bright dNK cells were sorted from the CD20− fraction with anti-CD56 microbeads (marked as tinted gate in CD20−CD56+ fraction). Shown is a representative example of postsort analysis (one of nine similar experiments is presented). Values are the percentage of CD45+ cells.

Overall the profile of cytokines secreted by the two dNK cell populations was similar, and clearly different from secretion by pNK cells (Fig. 7). Although pNK cells were characterized by high production of the inflammatory cytokines IL1β and TNF, only basal levels of these cytokines were generally detected in dNK cell supernatants, with CD56dim dNK cells producing the least amount. In addition, chemokines CCL3 (MIP-1α) and CCL4 (MIP-1β) were generally secreted at higher levels by pNK cells than by dNK cells, although the differences were not statistically significant. In contrast, dNK cells were characterized by higher secretion of CCL2 (MCP-1) and IL8.

FIG. 7.

FIG. 7.

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Secretion of selected cytokines, chemokines, and growth factors. CD56dim and CD56bright dNK cells, CD20−CD56− decidual MNL suspension (dNK cell-depleted fraction), or CD16+ pNK cells were plated into 96-well plates at 5 × 104/well ± IL15, 5 ng/ml (dNK), or IL2, 500 U/ml (pNK). Medium was collected after 24 h. Cytokines were measured using a Milliplex nonhuman primate cytokine kit. Only data from IL15/IL2-stimulated NK cells are shown. Box-and-whiskers histograms for the data are shown. The y-axes show the concentration of indicated cytokine in collected culture medium (pg/ml). Boxes represent the 25th and 75th percentile, bar in the box is the median, and whiskers are the minimum and maximum of all data. n = 6 for pNK cells, n = 9 for CD56bright or CD56dim dNK cells, n = 8 for dNK cell-depleted fraction (decT&Mph). Pairwise comparisons were performed using the nonparametric Mann-Whitney U-test, ***P < 0.001, **P < 0.01, *P < 0.05.

The analysis of growth factors also revealed the differences between cell populations. GM-CSF secretion by CD56bright and CD56dim dNK cells was elevated over pNK cells. Interestingly, the largest amounts of GM-CSF as well as of VEGFA were detected in the NK cell-depleted fraction of decidual MNL suspension. This fraction is enriched in T cells and macrophages, and macrophages are the likely producers of these growth factors [17]. We did not find any significant difference in the secretion of G-CSF among decidual leukocytes and pNK cells.

Despite the resemblance between CD56bright and CD56dim dNK cells in the secretion of the above-mentioned cytokines, the difference in IFNG production, which was revealed originally by intracellular staining analysis, was confirmed in secretion analysis. IFNG production by CD56dim NK cells was consistently higher than that by CD56bright dNK cells (Fig. 7). The possibility that the origin of IFNG in supernatants from CD56dim dNK cells could be from T cell contamination was ruled out because the medium from the NK cell-depleted fraction enriched with T cells had essentially no IFNG secretion (these low values can be seen on a log scale in Fig. 8A). Although some animals had a relatively low IFNG concentration in supernatants from CD56dim dNK cells, these numbers still exceeded their corresponding IFNG concentration in CD56bright cell supernatants several fold. Statistical analysis of paired samples within animals showed that IFNG values were significantly different between CD56bright and CD56dim dNK cells (Fig. 8A). A similarity to these IFNG results was revealed for IL6 secretion (Fig. 8A). Interestingly, IFNG was the only cytokine of all that were analyzed to be dependent on the presence of IL15 during culture (Fig. 8B) in all NK cell populations (for example, compare with IL6). Note that in pNK cells, the difference in IFNG secretion between cultures with and without IL2 was not statistically different.

FIG. 8.

FIG. 8.

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IFNG and IL6 secretion by decidual leukocytes. A) Individual data points from IL15 (dNK) and IL2-stimulated (pNK). The lines connect CD56bright and CD56dim dNK cells from individual animals, demonstrating consistently elevated IFNG and IL6 secretion by CD56dim cells, regardless of the within-animal relative secretion rate. **P < 0.01, Wilcoxon test (nonparametric, paired). B) Response to IL15 or IL2 treatment. INFG was the only cytokine in which secretion by dNK cells was responsive to the presence of IL15 in the culture media. As example of other cytokines, IL6 data are shown. Lines connect data points from individual animals. Wilcoxon test (nonparametric, paired) was performed to identify significant differences between dNK cell subtypes, as indicated on the plot. **P < 0.01.

DISCUSSION

In the present study we demonstrated the phenotypic and functional heterogeneity of dNK cells isolated from rhesus early pregnancy decidua. Two dNK cell populations specified according to their CD56 expression and other phenotypic and functional parameters, including surface receptor expression, activation, and proliferation and cytokine, chemokine, and growth factor secretion, were found to be distinct from pNK cells. A high prevalence of NK cells with a CD56bright phenotype matched the known predominance of CD56bright NK cells in the human decidua. However, functionally, the minor CD56dim NK population could be a key component of a maternal immune response to rhesus pregnancy, because these cells selectively express IFNG, in contrast to the CD56bright population. Intriguingly, individual animals seemed to have a characteristic proportion of dNK cell subsets and other leukocytes, across multiple pregnancies and regardless of sire, suggesting a genetic or physiological signature to decidual leukocyte trafficking and/or differentiation.

Phenotype and Activation of dNK Cells

CD56bright dNK cells are the prevalent immune cell in the rhesus decidua, as well as in human decidua [2, 18]. Previous human studies have revealed that CD56bright dNK cells have phenotypic and functional characteristics distinct from pNK cell subsets. There are reports that the human decidua contains a small proportion of CD56dimCD16+ NK cells [13, 10], but it is not clear if these cells are different from peripheral blood CD56dimCD16+ NK cells. Here we have demonstrated that in the rhesus monkey both CD56-expressing dNK cell subtypes, CD56bright and CD56dim dNK cells, are phenotypically and functionally distinct from CD56negCD16+/− pNK cells. Interestingly, rhesus CD56dim dNK cells were found to be mostly CD16-negative, whereas CD56bright dNK cells revealed high CD16 expression. The acquisition of the CD16 Fc receptor during the development of human NK cells is generally associated with the mature phenotype [19]. On the other hand, we found that the activation of dNK cells confirmed by CD69 expression results in loss of CD16 expression. Previously, we also defined elevated CD69 expression by CD56dim dNK cells in cynomolgus and vervet monkeys [20]. Additional differences between rhesus dNK cell subtypes were defined by perforin and CD20 staining. In CD56bright dNK cells, perforin was expressed at similar levels as in pNK cells, as previously shown in human studies [12], whereas rhesus CD56dim dNK cells revealed the lowest perforin expression. Interestingly, only CD56dim dNK cells demonstrated CD20dim staining. Dim expression of CD20 on a subset of CD16+/− pNK cells was previously reported [21] in the rhesus monkey. Our analysis of peripheral blood has confirmed this expression. Although a separate study will be required to reveal the biological function and significance of this expression, we showed that CD20dim expression in pNK cells is associated with a significantly higher level of NKp46 expression, but not of NKp30 or NKG2A/C.

We also defined NCR and NKG2A/C expression on rhesus dNK cells. Whereas NKG2A/C and NKp46 were expressed on all dNK subtypes, albeit at lower levels than on pNK cells, NKp30 expression was absent on CD56bright dNK cells, but NKp30 was moderately expressed on CD56dim and CD56neg dNK cells, in contrast to high expression on pNK cells. It is worth noting that divergent responses to the engagement of NKp46 and NKp30 in the total human dNK cell population have been reported [14]. Our discovery of differential expression of NKp46 and NKp30 on macaque dNK cells could help define a role these receptors may have in this unique tissue environment, and the diverse roles the dNK subsets may play in the decidua. Interestingly, an analogous NKp30-negative phenotype of NK cells was described earlier by Manaster et al. [22] as a unique characteristic of NK cells in the human cycling endometrium (eNK cells). These authors suggested that eNK cells are immature and inactive cells awaiting pregnancy. In addition, analysis of the gene expression profile of eNK and dNK cells revealed that they are distinct subsets and both differ from pNK cells [23]. We have now shown that MKI67 is highly expressed in most CD56bright dNK cells and a substantial proportion of CD56dim dNK cells, but few CD56neg dNK or pNK cells. The signals in the decidua that promote dNK proliferation remain to be further investigated, but high MKI67 expression may point to the possibility that CD56bright dNK cells may be an immature population in this mucosa, and perhaps the CD56dim cells are derived from the CD56bright population.

Cytokines and Growth Factors

Although we recognize that in vitro secretory activity may not fully mirror in vivo function, it is an appropriate starting point for obtaining data for formulating hypotheses and designing future studies to test in vivo function. Compared to pNK cells, the current data demonstrate that the decidua is a region of selective cytokine, chemokine, and growth factor expression and secretion. First, there was low or restrained IL1B, TNF, and IL6 secretion by dNK cells. These data indicate that the decidua of pregnancy is an environment of distinctly suppressed inflammatory potential, at least with regards to NK cell secretion. Conversely, a number of cytokines, growth factors, and chemokines were significantly elevated in dNK cells compared to cells from the peripheral blood. CSF2 (GM-CSF) was moderately elevated in the decidua, especially so in T cells and macrophages. We have previously shown that CSF3 (G-CSF) is important in the functional differentiation of peripheral blood monocytes to macrophages in vitro [24], and in the decidual environment, perhaps CSF2 may serve a similar role. This suggests that dNK cells (and perhaps T cells and resident macrophages themselves) are important in the maturation of additional decidual macrophages, which increase dramatically during the first weeks of gestation [25–27]. In addition to these growth and differentiation factors, the potent chemokine CCL2 (previously known as MCP-1) was strongly increased in the decidua compared to pNK cells. CCL2 is an important regulator of monocyte and macrophage chemotaxis and suggests that dNK cells may play a role in the influx of these cells to the decidua in early pregnancy. This is also in agreement with a positive correlation shown between the abundance of dNK and decidual macrophages. Moreover, there was a significant elevation of IL8 in decidual cells vs. peripheral blood cells. IL8 is a potent chemoattractant for not only neutrophils but also trophoblasts at the implantation site [9]. Influx of neutrophils to the implantation site may be beneficial to pregnancy in the phagocytosis of the regressing epithelial plaque in the rhesus implantation site, or cellular debris from maternal spiral arterioles undergoing remodeling at this time.

The role of decidual leukocytes in the regulation of angiogenesis and vascular development remains incompletely understood. Although human dNK cells have been reported to secrete VEGFA when 48- or 72-h culture supernatants were tested [8, 9, 28], only low levels of VEGFA were detected in rhesus dNK cell 24-h supernatants. Manaster et al. [22] reported that human eNK cells, in contrast to dNK cells, failed to secrete VEGFA (and placental growth factor). One possibility is that not all rhesus VEGFA isoforms were detected in the Luminex assay employed in the current study. Further analysis of the VEGFA isoforms secreted by rhesus NK cells is warranted. It is also possible that rhesus dNK cells may secrete different amounts of VEGFA at different times in gestation, because we evaluated only cells from ∼Day 36 of gestation in the current study.

Our data also show that decidual macrophages might be equivalent in their capacity to produce VEGFA, although quite variable. There is a close association in the rhesus implantation site between macrophages and decidual spiral arterioles in particular [25]; thus, both dNK cells and decidual macrophages are well positioned to contribute to vascular regulation in early pregnancy. Lash et al. [29] have reported that in comparing human dNK cell supernatant versus unfractionated decidual cell supernatant (from 8 to 10 wk of gestation), the latter actually did increase extravillous trophoblast invasion with in vitro assays to a greater extent than the former. Thus, both NK and monocyte-derived cells are likely to contribute significantly to the immune dialog between the decidua and the placenta.

In the present study we revealed that the minor CD56dim dNK cell population in early pregnancy rhesus decidua is characterized with an activated phenotype, and produces IFNG and IL6, in contrast to the major CD56bright dNK cell population. The secretion of IFNG is interesting in that an important role in pregnancy has been demonstrated in mice [30]. NK cell-deficient mice exhibited abnormal placentation with hypocellularity or necrosis of the decidua and deficient remodeling of the maternal spiral arteries. It was demonstrated that IFNG is the key dNK cell-derived factor relevant to these outcomes, although the precise pathway of IFNG signaling in the mouse implantation site is not known. IFNG production by human dNK cells has also been reported [22, 31, 32] and a role of IFNG controlling trophoblast invasion by inducing apoptosis in extravillous trophoblasts [32] and down-regulation of trophoblast metalloprotease-2 and -9 [32, 33] has been proposed. Basal IFNG production by freshly isolated human dNK cells was shown to be low [6, 34], but was detected after PMA and ionomycin stimulation [35] or in the total decidual leukocyte population [22]. The analysis of a CD56-positive human decidual lymphocyte population at a single-cell level (ELISPOT, immunohistochemistry) also indicated that the proportion of IFNG-positive cells within the CD56+ cell population was not high [32, 36]. Thus, the precise source and role for IFNG in the human or nonhuman primate remains to be further elucidated.

dNK Cells in Repeated Pregnancies

It is likely that the role of dNK cells in pregnancy-related pathologies, including recurrent pregnancy loss and pregnancy complicated by preeclampsia or intrauterine growth restriction, is multifactorial, and involves interaction with decidual macrophages, dendritic cells, and T cells, as well as signaling to trophoblasts. Here were observed that a higher proportion of CD56dim dNK cells or a lower proportion of CD56bright dNK cells is correlated with an increased number of T cells in the decidua. An appropriate in vivo model will be important for discerning NK cell importance in pregnancy. To this end, it was intriguing that our analysis of more than one pregnancy in an individual animal let us compare her decidual immune cell profile in different pregnancies. It was discovered that the proportion of various immune cells in the decidua is an individual characteristic that may be predictably reproduced in future pregnancies. The causes of conditions like recurrent miscarriage are not fully understood, and it is possible that variable individual decidual immune cell profiles may provide a paradigm for identifying animals at risk for pregnancy loss, and stratifying their responses to pregnancy stressors. Although we do not know at this time what may be the elements that contribute to the individual profiles of decidual leukocytes, some possibilities include polymorphisms in chemotaxis, adhesion, and homing of these leukocytes to the decidua. Moreover, there are likely to be individual variations in the levels of secretion of cytokines, chemokines, and growth factors. These may determine the number of immunoregulatory cells in this unique microenvironment that will be required to reach adequate levels of secreted factors. Identification of strategies to predict the decidual environment, and perhaps modify this environment, may provide for translational opportunities in the treatment of infertility and pregnancy loss.

Supplementary Material

Supplemental Tables S1 and S2
Click here for additional data file. (476.7KB, pdf)

ACKNOWLEDGMENT

We thank the Veterinary, Immunology, Centralized Protocol Implementation, Pathology, and Assay Service staff of the WNPRC for procedures with animals and hormone assays to confirm ovulation and pregnancy, and for flow cytometry and tissue collection support. We are grateful to Songwon Seo and Richard Chappell of the Department of Biostatistics and Medical Informatics for statistical consulting and assistance.

Footnotes

1

Supported by National Institutes of Health grants RR21876 and AI076731, and American Recovery and Reinvestment Act grants HD37120-06A2 and HD053925-01S1 to T.G.G. and RR00167 to the Wisconsin National Primate Research Center. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program grant numbers RR15459-01 and RR020141-01. This publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.

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Associated Data

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Supplementary Materials

Supplemental Tables S1 and S2
Click here for additional data file. (476.7KB, pdf)

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