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Biol Reprod 2006, 10.1095/biolreprod.106.052654
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BIOLOGY OF REPRODUCTION 75, 562–567 (2006)
DOI: 10.1095/biolreprod.106.052654
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Effector Activity of Decidual CD8+ T Lymphocytes in Early Human Pregnancy1

Paula J. Scaife 3, Judith N. Bulmer 4, Stephen C. Robson 3, Barbara A. Innes 3, and Roger F. Searle 2 5

Schools of Surgical and Reproductive Sciences,3 Clinical and Laboratory Sciences,4 Medical Education Development,5 University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, Tyne and Wear, NE1 4LP, United Kingdom

ABSTRACT

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7–14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR32] of 32.7 ± 5.8) and against K562 target cells (mean SCR32 of 20.3 ± 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8+ T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.

cytokines, decidua, immunology, trophoblast

INTRODUCTION

In early human pregnancy, approximately 30%–40% of stromal cells in the decidualized endometrium (decidua) are leukocytes; CD56brightCD16– uterine natural killer (uNK) cells and CD14+ macrophages predominate, with CD3+ T lymphocytes accounting for fewer than 20% of the leukocyte population [1–3]. Unlike in peripheral blood, there are more CD8+CD3+ T cytotoxic cells present in decidua than CD4+CD3+ T helper cells [4]. The number of CD3+ T cells in the uterus remains constant during the menstrual cycle and pregnancy, leading to the suggestion that T lymphocytes, while providing immune protection against invading pathogens, are unlikely to play a key role in the establishment and maintenance of pregnancy [4].

Other investigators, however, have shown that CD8+ T cells in the nonpregnant human endometrium are capable of cytolysis, with a substantial reduction in cytolytic ability in the secretory phase of the menstrual cycle [5], suggesting that the loss of cytolytic potential of uterine CD8+ T lymphocytes could prevent rejection of the implanting conceptus. Moreover, Klentzeris et al. [3] reported an increase in CD8+ T lymphocyte numbers around the time of implantation (Days 4 and 7 after ovulation, assessed by the luteinizing hormone surge) and fewer CD8+ T cells in the endometrium of women with unexplained infertility [6], indicating a possible role for CD8+ T lymphocytes in implantation.

Decidual CD8+ T lymphocytes may be involved in the maintenance of pregnancy via the production of cytokines [7]. Although successful pregnancy is associated with a predominant Th2 type cytokine profile, with Th1 type cytokines being considered detrimental to pregnancy [7–13], emerging evidence suggests that the role of the Th1:Th2 balance in determining pregnancy outcome may be less clear cut [14–16]. The contribution to the local intrauterine cytokine profile made by decidual CD8+ T lymphocytes is largely unknown [7].

For successful pregnancy, invasion of the uterine decidua by extravillous trophoblast is crucial [17]. The mechanisms that control trophoblast invasion into the uterus are incompletely understood, but cytokines have been shown to regulate trophoblast growth, differentiation, and invasion; first trimester trophoblast cells treated with the Th2 type cytokines interleukin (IL) 6 and IL10 proliferated, whereas cell numbers and invasive properties were reduced in the presence of transforming growth factor (TGFB) 1, 2, and 3; tumor necrosis factor (TNF); and interferon gamma (IFNG) [18–20]. The ability of decidual CD8+ T lymphocytes, however, to affect trophoblast invasion is unknown.

The aim of the present study was to characterize the effector properties of decidual CD8+ T lymphocytes in normal early human pregnancy, and to assess whether decidual CD8+ T lymphocytes via their secretory products could affect trophoblast invasion.

MATERIALS AND METHODS

Tissues

After approval from the Newcastle and North Tyneside Joint Ethics Committee and appropriate informed consent was obtained, early pregnancy decidua (7 wks gestational age [GA] calculated from ultrasound measurement of crown rump length, n = 1; 8 wks GA, n = 15; 9 wks GA, n = 5; 10 wks GA, n = 11; 11 wks GA, n = 4; 12 wks GA, n = 11; 13 wks GA, n = 3; 14 wks GA, n = 1) were obtained from women admitted to the Royal Victoria Infirmary, Newcastle-upon-Tyne, for surgical termination of pregnancy under Clause E of the 1969 Abortion Act. Decidual tissue was selected macroscopically by its opaque gray-white solid appearance and separated from fetal and placental tissue. Placental tissue was identified by its branching fronded appearance.

Decidual Cell Suspensions

Decidual single cell suspensions were prepared as described previously [21], adjusted to 1x106/ml in complete medium (RPMI 1640 supplemented with 10% fetal calf serum [FCS], 1000 U/ml penicillin, 1 mg/ml streptomycin, and 2 mmol/l L-glutamine) (Sigma-Aldrich, Poole, UK), and cultured for 12 h at 37°C in 5% CO2 to remove adherent cells. For redirected cytotoxicity assays, decidual cell suspensions were supplemented with 100 U/ml recombinant human IL2 (Peprotech EC Ltd, London, UK) before overnight incubation [5].

Positive Selection of CD8-Positive Lymphocytes

Following overnight incubation, CD8-positive lymphocytes were positively selected from the nonadherent decidual cells. Decidual cells were first incubated with anti-CD8 murine monoclonal antibody (clone DK25; DAKO, Cambridge, UK), followed by anti-mouse immunoglobulin G-coated magnetic microbeads (MACS; Miltenyi Biotech Ltd, Surrey, UK); CD8+ cells were positively selected using a MidiMACS magnetic separation system following the manufacturer's protocol. Purity was routinely confirmed by either flow cytometry or immunohistochemistry on cell smears. For flow cytometry, purified cells were treated with CD8 fluorescein isothyocyanate/CD56 phycoerythrin/CD3 peridinin chlorophyll-A protein-cytochrome 5.5 combination antibody (BD Biosciences, Aalst, Belgium). For immunohistochemistry, cell smears were labeled using a modified avidin-biotin complex (ABC) method using the Vectastain Elite kit (Vector Laboratories, Peterborough, UK). Briefly, the smears of positively selected cells (already incubated during selection with anti-CD8 antibody) were incubated sequentially with appropriately diluted biotinylated horse anti-mouse immunoglobulins (Vector Laboratories; 30 min) and the Vectastain ABC-peroxidase reagent (Vector Laboratories; 30 min). The reaction was developed using NovaRed (Vector Laboratories) to give a red reaction product. Smears were lightly counterstained with Mayer hematoxylin, blued in Scott tap water, dehydrated, cleared, and mounted in DPX (BDH).

Cytotoxicity Assays

Decidual CD8+ effector cell cytotoxic function was assayed either in a standard 4-h chromium–51 (51-Cr) release assay against the human erythroleukemia cell line K562 target cells to assay NK cell-sensitive cytotoxicity, as described previously [22], or in a CD3 antibody redirected chromium release assay against the murine mastocytoma Fc{gamma} cell line P815 target cells that require T-cell receptor signal transduction to mediate cytotoxicity [5]. For the redirected chromium release assay, decidual CD8+ T cells were assessed at 7 wks GA (n = 1), 8 wks GA (n = 2), 9 wks GA (n = 1), 10 wks GA (n = 3), 12 wks GA (n = 1), 13 wks GA (n = 2), and 14 wks GA (n = 1); and for the standard chromium release assay at 8 wks GA (n = 2) and 13 wks GA (n = 1). The K562 and P815 cell lines were a kind gift from Dr. Colin Brooks, University of Newcastle-upon-Tyne. 51-Cr-labeled P815 cells were preincubated in 10 µl/ml CD3 murine monoclonal antibody (clone UCHT1, DAKO) for 30 min at 37°C in 5% CO2. Labeled target cells (1x104 in 100 µl complete medium) were incubated with a range of doubling dilutions of decidual CD8+ effector lymphocytes to give final effector:target (E:T) ratios of between 60:1 and 1:1 in triplicate in a final volume of 200 µl in round-bottomed plates. The highest E:T ratio was determined by the yield of decidual effectors. The plates were centrifuged at 200 g for 10 sec, incubated at 37°C in 5% CO2 for 6 h, centrifuged at 200 g for a further 10 sec, and then 100 µl of each supernatant was harvested and counted in a gamma counter to determine isotope release. Minimum and maximum 51Cr release was determined in 10% FCS and 30% detergent solution (Hospec General Purpose Neutral liquid detergent; Youngs Detergents, Cheshire, UK), respectively. Specific lysis was determined as described previously [22]. The % Specific Chromium Release (%SCR) at each E:T ratio was subjected to linear regression analysis (Microsoft Excel; Microsoft Corporation, Redmond, WA), and the mean %SCR (± SEM) at each E:T ratio for all the samples was then calculated. In order to allow comparison between the CD3 antibody redirected and the standard chromium release assays, the results are also presented as the % Specific Chromium Release at E:T ratio of 32:1 (%SCR32).

Induction of Cytokine Production by CD8+ Decidual Lymphocytes

Positively selected CD8+ decidual lymphocytes were resuspended in complete medium (1x106 cells/ml), and 100 µl of cell suspension was dispensed into wells of round-bottomed 96-well plates. An equal volume of phytohemagglutinin-P (PHA-P; 10 µg/ml) (Sigma-Aldrich) was added to stimulate CD8+ decidual lymphocytes. Plates were incubated for 24 and 48 h. After this period, supernatants were collected, spun at maximum speed in a microcentrifuge (Eppendorf) for 2 min, and cell-free supernatants were stored at –70°C.

Multiplex Cytokine Analysis

Production of granulocyte-macrophage colony-stimulating factor (CSF2), IFNG, IL1B, IL2, IL4, IL6, IL8, IL10, IL12, and TNF by decidual CD8+ lymphocytes was analyzed in culture supernatants using the Beadlyte human multi-cytokine detection system 3 (Upstate, Milton Keynes, UK) following the manufacturer's protocol. The sensitivity of the multiplex kit used was 1 pg/ml for each cytokine assayed apart from IL12 (2 pg/ml) and IFNG (16 pg/ml). Supernatants were analyzed in duplicate. Cytokine analysis was performed using the Luminex 100 integrated system 2.2, and results were analyzed using Luminex 100 IS software SP1.

Invasion Assays

The effect of decidual CD8+ T-lymphocyte culture supernatants on the ability of extravillous trophoblast cells from placental explants to invade through Matrigel was assessed using invasion assays, as described previously [20]. Briefly, chorionic villous tips were minced to approximately 0.5 mm3 and resuspended in 500 µl DMEM-F12 medium containing 10% FCS, penicillin/streptomycin, and amphotericin B, such that 15 µl constituted approximately 10 mg of tissue. Fifteen microliters of placental tissue explant suspension were pipetted onto Matrigel-coated 8-µm pore Falcon filters, 200 µl DMEM-F12 medium was added to the lower chamber of each well, and the explants were incubated overnight (37°C in 5% CO2 in air). The following day (Day 1), a further 200 µl complete medium was added to the bottom chamber of each well and 200 µl CD8+ cell culture supernatant (48-h conditioned medium) was dispensed into the upper chamber of each well, ensuring that villous explants were covered in medium. Control wells consisted of placental explants cultured in the presence of 200 µl PHA-P (10 µg/ml) containing DMEM-F12 medium in the upper chamber.

Test CD8+ cell supernatant (n = 16) and control (n = 6) cultures were set up in duplicate. Plates were then cultured at 37°C in a 5% CO2 incubator for 7 days. After 7 days' incubation, the Matrigel and explants were removed, and the upper side of the membrane was cleaned with a cotton swab. In order to visualize and quantify the number of invaded cells, the filters were fixed in 10% neutral buffered formalin, stained with hematoxylin and eosin, and mounted on glass microscope slides using synthetic mountant. The total number of cells on the underside of the filter was counted at x100 magnification for the entire area of the filter. Since there was considerable variation between different placental explants, mean cell counts for each invasion assay were normalized with respect to PHA-P (control) values for that assay, as previously described [20].

Statistical Analysis

Descriptive statistical analysis and assessment of normality was performed using Prism (GraphPad Software Inc.). Except where indicated, data showed a normal distribution. For the cytotoxicity assays, unpaired Student t-tests were used. For the invasion assays, paired Student t-tests were used. P values < 0.05 were considered statistically significant.

As levels of the cytokines in both 24- and 48-h supernatants were not normally distributed, the Mann-Whitney U-test was used to determine whether cytokine levels differed between 24- and 48-h supernatants. As cytokine levels did not differ between different culture periods, only 48-h supernatants were used for statistical analysis, which was performed with one-way analysis of variance to determine whether cytokine production varied with GA. Those cytokines that changed with GA were then subjected to unpaired Student t-tests with Bonferroni correction, the level of significance being lowered to P < 0.0125 to take into account the multiple analyses.

RESULTS

Phenotype of Positively Selected Decidual CD8+ Lymphocytes

In order to maximize the number of cells available for functional assays, immunohistochemistry for CD8 was routinely performed on cell smears to check purity. Using cell smears, 95% ± 1% (mean ± SEM) of decidual lymphocytes positively selected by immunomagnetic separation were CD8+ cells (n = 30). The efficiency of positive isolation using MidiMACS was confirmed using flow cytometry (Fig. 1). CD3+ cells comprised >96.3% of the purified fraction. CD56+CD3– cells (presumed to be NK cells) were either absent or comprised less than 1.0% of the fractions, although CD8+CD56+ T cells were present (Fig. 1).


Figure 1
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  		FIG. 1. Flow cytometry histograms obtained for CD3/CD56, CD8/CD56, and CD3/CD8 labeling of decidual lymphocytes obtained in the purified fraction following CD8+ MidiMACS

Cytolytic Activity of Decidual CD8+ Lymphocytes

CD8+ decidual lymphocytes from normal early pregnancy samples exhibited cytolytic activity against P815 target cells. The results for all samples are summarized in Figure 2. The mean %SCR32 for normal pregnancy samples (n = 11) was 32.7 ± 5.8. With the exception of one sample at 12 wks GA, which had a mean %SCR32 of 80.1, the cytolytic activity of decidual CD8+ lymphocytes did not appear to significantly vary between the 7- to 14-wk GA range (data not shown); but due to the small subject numbers for each GA week, statistical analysis was not performed. Decidual CD8+ lymphocytes also exhibited cytolytic activity against K562 cells in a standard chromium release assay (n = 3). The mean %SCR32 was 20.3 ± 0.5 (Fig. 2). There was no difference in cytolytic ability between the redirected chromium release assay and the standard chromium release assay.


Figure 2
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  		FIG. 2. Summary of cytolytic activity of CD8-positive lymphocytes from normal early pregnancy decidua against P815 target cells (NP, n = 11) and K562 cells (K, n = 3). Data are subjected to linear regression analysis and expressed as mean %SCR ± SEM of total samples assayed for each E:T ratio

Cytokine Production by Decidual CD8+ T Lymphocytes

Detectable levels of CSF2, IFNG, IL1B, IL2, IL6, IL8, IL10, IL12, and TNF were found in decidual CD8+ T lymphocyte culture supernatants after incubation of 24 (n = 21) and 48 (n = 21) h. Levels of IL4 fell below the range of the Luminex assay. As cytokine levels in supernatants did not differ between 24- and 48-h incubation samples (data not shown) for any of the cytokines examined, the cytokine levels from 48-h supernatants were used for analysis of GA-associated changes in cytokine production.

Levels of CSF2, IL1B, IL2, IL10, IL12, and TNF did not vary among the GA groups examined (Fig. 3). Levels of IL6 from the 11-wk group were higher than in the other GA supernatants, but this did not reach significance. Both IL8 and IFNG were detected at high levels in all decidual CD8+ T lymphocyte supernatants with no GA changes (Fig. 3).


Figure 3
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  		FIG. 3. Comparison of cytokine levels in decidual CD8+ T-lymphocyte cell-free culture supernatants from different GA groups (8 wk, n = 10; 9 wk, n = 8; 10 wk, n = 8; 11 wk, n = 8; 12 wk, n = 8). Graphs show cytokines produced in high (A) and low (B) amounts. Data are expressed as mean pg/ml of duplicates ± SEM

Effect of Decidual CD8+ T Lymphocyte Culture Supernatants on Trophoblast Invasion

Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel when compared with the PHA-P control (n = 16, mean number of invaded cells normalized to PHA-P ± SEM: 280 ± 61.3 vs. 100 ± 0, P = 0.006; Fig. 4). When the PHA-P control was compared with medium-alone controls, there was no difference in the number of invaded extravillous trophoblast cells (data not shown). When analyzed by GA, supernatants from 8-wk samples increased invasion (n = 6, 263 ± 54.5) compared with the PHA-P control (100 ± 0, P = 0.001; Fig. 4). Decidual CD8+ T lymphocyte supernatants from 10- (n = 4) and 12-wk (n = 6) GA samples also showed a trend to increase extravillous cytotrophoblast cell invasion compared with the PHA-P control (data not shown), but this did not reach statistical significance.


Figure 4
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  		FIG. 4. Effect of decidual CD8+ T-lymphocyte cell-free culture supernatant on the invasive capacity of extravillous cytotrophoblast cells derived from placental explants. Data are expressed as mean normalized to PHA-P control ± SEM

DISCUSSION

This study demonstrates for the first time that decidual CD8+ T cells from normal early pregnancy are functionally capable of cytolytic function and represent a potential source of cytokines that could affect trophoblast invasion. The present observation of cytolytic activity of decidual CD8+ T cells during early pregnancy extends previous studies in the nonpregnant uterus [5], all previous reports of decidual cytolytic function having focused on uNK cells [22–26]. The sustained cytolytic capacity of decidual CD8+ T cells throughout the first trimester of pregnancy suggests that these cells are an important functional component of the decidual immune cell population, and may play a role in pregnancy maintenance.

Recently distinct CD8+ T lymphocyte subsets have been recognized to display differential cytolytic ability [27–30], with CD8+CD56+ T cells having upregulated cytolytic activity against K562 cells compared with CD8+CD56– T cells [29]. Although the relative contribution of the different decidual CD8+ T-cell subsets to the overall cytotoxicity is unknown, flow cytometry with triple CD3/CD8/CD56 labeling demonstrated that the cytotoxicity activity reflected only the presence of the decidual CD8 T cells as there was less than 1% contaminating CD3–CD56+ NK cells.

Although cytolytic in vitro, it is unknown whether decidual CD8+ lymphocytes lyse extravillous trophoblast cells in vivo. Decidual CD8+ lymphocytes could, however, recognize human leukocyte antigen (HLA)-C expressed by extravillous trophoblast cells [31]. Recognition of HLA-C by maternal immune cells may be important for normal pregnancy, especially as women lacking killer activating receptors that interact with HLA-C are more likely to suffer from preeclampsia [32], while women lacking killer inhibitory receptors are more likely to suffer from recurrent miscarriage [33]. It is therefore possible that recognition by decidual CD8+ T lymphocytes of HLA-C expressed by trophoblasts may result in the generation of a cytotoxic response that regulates the extent and depth of trophoblast invasion. Other investigators [34] have reported that human placental trophoblast cells can activate CD8+ regulatory T cells that require costimulation through a member of the carcinoembryonic antigen family present on early gestation trophoblasts.

Decidual CD8+ lymphocytes produced much greater amounts of IFNG and IL8 than the other eight cytokines assayed. These findings, together with the undetectable levels of IL4 and low levels of IL10, support the suggestion that normal pregnancy is not simply a Th2 type phenomenon [15]. Production of IFNG by decidual CD8+ T lymphocytes may represent an additional mechanism by which trophoblast invasion and growth is regulated. Although IFNG has been shown to be essential to maintain decidual integrity in murine pregnancy [35], we have recently demonstrated that the invasive capability of human first trimester extravillous trophoblast cells was reduced by IFNG [19]. The high levels of IFNG may have had secondary cytokine effects as the low IL10 levels in decidual CD8+ T lymphocyte cell-free culture supernatants may reflect the suppressed release of IL10 by IFNG [36, 37]. Since IL10 enhances NK cell cytotoxicity [38], low levels of this cytokine in normal pregnancy may serve to downregulate uNK cell cytotoxicity locally at the implantation site. The high levels of IL8 suggest that this cytokine may play a hitherto unappreciated role in the maintenance of pregnancy owing to its angiogenic or chemoattractant properties. IL8 has been reported to increase production of both matrix metalloproteinase (MMP) 2 and MMP9 by endothelial cells [39]; this would suggest that this cytokine may be important for the vascular remodeling required for the successful establishment of the placenta. Other researchers [40] have reported abundant levels of IL8 mRNA and protein during endometrial receptivity and early pregnancy, raising the possibility that production of this chemokine may serve as an important regulator of decidual leukocyte recruitment needed to modulate trophoblast invasion.

The overall stimulatory effect of cytokines and other factors produced by decidual CD8+ T lymphocytes on trophoblast invasion raises the possibility that CD8+ lymphocytes could play a role in the regulation of trophoblast function. Since IFNG, TNF, and TGFB1–3 inhibit invasion of extravillous trophoblast in vitro [19, 20], the presence of other cytokines in decidual CD8+ T-lymphocyte culture supernatants are likely responsible for the increase in extravillous trophoblast cell invasion. Candidate cytokines include IL1B [41, 42] and IL6 [43]. Whether IL8 has a stimulatory effect on trophoblast invasion is unknown. Although receptors are expressed on extravillous cytotrophoblast for CSF2, IFNG, IL10, and TNF [44–47], the status of CXCR1 expression for IL8 is unknown. Previous studies have identified individual cytokines that can regulate trophoblast function [19, 20, 41, 43, 48], but the present study represents the first to examine trophoblast invasion within a complex cytokine environment that is more representative of the fetal-maternal interface where cytokines and growth factors interact.

In summary, decidual CD8+ T lymphocytes in normal early pregnancy are capable of both cytolysis and cytokine production. High levels of the Th1 type cytokines IFNG and IL8 and low levels of the Th2 type cytokines IL4 and IL10 indicate that these cells do not evoke a predominant Th2 type cytokine environment. Decidual CD8+ T lymphocytes can regulate the invasive capacity of trophoblast cells in vitro. We speculate that decidual CD8+ T lymphocytes may regulate trophoblast function and hence contribute to placental development.

ACKNOWLEDGMENTS

The authors wish to acknowledge the staff of the Royal Victoria Infirmary, Newcastle-upon-Tyne, for their assistance in sample collection.

FOOTNOTES

1 Supported by the Dr. William Edmund Harker Foundation, RVI, Newcastle-upon-Tyne, and the BBSRC (S19967). Back

2 Correspondence: Roger F. Searle, Anatomy and Clinical Skills, School of Medical Education Development, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, United Kingdom. FAX: 44 191 222 5517; R.F.Searle{at}ncl.ac.uk Back

Received: 22 March 2006.

First decision: 28 April 2006.

Accepted: 16 June 2006.

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