Decidual T cells exhibit a highly differentiated phenotype and demonstrate potential fetal-specificity and a strong transcriptional response to interferon

Abstract

Immune tolerance during human pregnancy is maintained by a range of modifications to the local and systemic maternal immune system. Lymphoid infiltration is seen at the implantation site of the fetal–maternal interface and decidual Natural Killer (NK) cells have been demonstrated to facilitate extravillous trophoblast (EVT) invasion into maternal decidua during the first trimester, optimising haemochorial placentation. However, although there is considerable T cell infiltration of the maternal decidua the functional properties of this T cell response remain poorly defined.

We investigated the specificity and regulation of CD4+ and CD8+ T cells obtained from human third trimester decidua and demonstrated that decidual CD4+ and CD8+ T cells exhibit a highly differentiated effector memory phenotype in comparison to peripheral blood and display increased production of interferon-γ (IFN-γ) and interleukin 4 (Il-4). Moreover, decidual T cells proliferated in response to fetal tissue, and depletion of T regulatory cells led to an increase in fetal-specific proliferation. HY-specific T cells were detectable in the decidua of women with male pregnancies and were shown to be highly differentiated. Transcriptional analysis of decidual T cells revealed a unique gene profile characterized by elevated expression of proteins associated with the response to interferon signalling.

These data have considerable importance both for the study of healthy placentation and for the investigation of the potential importance of fetal-specific alloreactive immune responses within disorders of pregnancy.

Introduction

Successful pregnancy in eutherian placental mammals requires maternal immunological tolerance of the developing semi-allogenic fetus (1). Improved understanding of the physiological mechanisms that underlie this immune regulation is likely to provide insights into the etiology of pregnancy complications and may impact on studies of immunological tolerance in the setting of transplantation and cancer.

The classical model of immune regulation during human pregnancy has been based upon a relative shift in the maternal immune response from an inflammatory Th1-cytokine pattern to a Th2 profile (2). However, it is now recognized that the complex immunological interactions at the maternal interface cannot be explained with this simple binary classification. Indeed, villous implantation and invasion (3) are facilitated by an inflammatory environment and maternal T-cell function exhibits great diversity and plasticity (4).

The formation of the human hemochorial placenta, involves the invasion of fetal extravillous trophoblast (EVT) cells which remodel maternal spiral arteries and reduces their resistance to blood flow. Prior to and during this process, the maternal uterine endometrium is transformed into a layer termed the decidua (5). In early pregnancy the decidua accumulates large numbers of specialized uterine (u) NK cells (CD56^bright CD16-) and their interaction with EVT plays a key role in successful placentation (6,7). However, as pregnancy progresses the number of uNK cells declines and by the third trimester T-lymphocytes become the predominant leukocyte population (8). Despite this, the role that decidual T cells play in modulating the uterine environment, and their potential recognition of the fetus, remain controversial questions.

Maternal T cells within decidua are likely to make direct anatomical interactions with EVT and would therefore become exposed to fetal antigen. However, EVT does not express HLA-A, HLA-B or HLA class II alleles, although it does retain HLA-C expression (9) and significantly higher levels of activated T cells and T regulatory cells are induced within decidua of HLA-C mismatched pregnancies (10). Murine models suggest that fetal proteins are presented to the maternal immune system indirectly, by maternal antigen presenting cells (11). Indeed, large numbers of fetal trophoblast cells (and fragments) are shed into the maternal circulation during ‘normal’ pregnancy and provide a rich supply of fetal and placental antigens to the maternal immune system. Conversely, maternal dendritic cells appear to be limited in their ability to migrate from the pregnant uterus (12) and epigenetic silencing of key chemokines in the decidual stroma may limit T cell access to the decidua (13).

In human pregnancy maternal CD8+ T cells with specificity for fetal antigens are detectable in maternal peripheral blood both during (14) and after (15, 16,17) pregnancy. Studies of T cell biology directly within human decidua are more limited and the antigenic specificity of these cells is unclear. Effector memory CD8+ cells have been demonstrated in this setting and shown to express low levels of perforin and granzyme (18).

The mechanisms by which decidual T cells are regulated are unclear and may depend on the potential of these cells to gain anatomical access to fetal tissue. Large numbers of T regulatory cells are generated during pregnancy (19,20) and murine studies show that these are essential for fetal survival (21). Inhibitory checkpoint proteins such as PD-1, T Cell Immunoglobulin and Mucin-Domain Containing 3 (Tim-3) play a critical role in murine models of transplantation and pregnancy (22). Antibody-mediated blockade results in allograft and fetal rejection (23,24). Expression of these inhibitory checkpoint proteins has been demonstrated on human decidual CD8+ T cells (25) whilst Programmed Death Ligand 1 (PD-L1) is also expressed in human decidua (22, 26).

The objectives of this study were to perform a detailed phenotypic and transcriptional assessment of both decidua-derived CD4+ and CD8+ T cells in order to comprehensively characterise these tissue derived subsets and their antigenic specificity. Our findings show that decidua is enriched with activated effector T cells which demonstrate an unusual transcriptional profile dominated by interferon responsive pathways. Moreover effector T cells within decidua have the potential to recognise fetal tissue and this functional response is actively suppressed by local T regulatory cells.

Methods

Mononuclear cell isolation from placental tissue and peripheral blood

In total N=90 placentae was obtained from healthy mothers undergoing 3^rd trimester elective caesarean section. Peripheral blood and cord blood samples were also obtained at this time. Decidua basalis was dissected and dissociated via Miltenyi GentleMACs and enzymatically digested using 1mg/ml collagenase (Sigma-Aldrich, UK) and 200U/ml of DNAse (Sigma-Aldrich, UK) at 37°C for 30 minutes. Digested tissue was then filtered, washed using RPMI (Sigma-Aldrich, UK) and mononuclear cells were isolated using density gradient medium Lymphoprep (Alere, UK). Maternal and cord blood mononuclear cells were similarly separated using density gradient.

Phenotyping of maternal and decidual T-cells using flow cytometry

To phenotype T cell memory status, 1 x 10⁶ fresh maternal or decidual mononuclear cells (N= 45) were stained with: CD3 Amcyan (clone SK7, Becton Dickinson, UK); CD4 PE (clone RPA-T4, Cambridge Bioscience, UK); CD8 Efluor450 (clone OKT8, eBiosciences, UK); CD45RA AF700 (clone HI100, Biolegend, UK); CCR7 FITC (clone 150503, Becton Dickinson); CD27 APCeFluor780 (clone 0323, eBiosciences, UK); CD28 PerCPCy5.5 (clone L293, Becton Dickinson, UK); CD57 APC (clone HCD57, Biolegend, UK); CD14 ECD (clone RM052, Beckman Coulter, UK); CD19 ECD (clone J3-119, Beckman Coulter, UK). Samples were then analysed using LSRII (Becton Dickinson, UK).

To phenotype intracellular cytokines and T regulatory cells, prior to antibody staining, fresh maternal and decidual monocular cells (N=35) were incubated in RPMI supplemented with L-glutamin (Sigma-Aldrich, UK), bovine foetal calf serum (Sigma-Aldrich, UK), and 50ng/ml of PMA (Sigma-Aldrich, UK), 1ug/ml of Ionomycin (Sigma-Aldrich, UK) and 1.25ug/ml of monensin (Sigma-Aldrich, UK) was added for 4 hours. Unstimulated samples were set up in parallel. Samples were surface stained with: CXCR3 PECy7 (clone IC6, Becton Dickinson, UK); CD25 FITC (clone IM0478U, Beckman Coulter, UK); CD127 Efluor 780 (clone ebioRDR5, eBiosciences, UK); CD8 BV510 (clone RPA-T, Biolegend, UK); CD4 PerCP Cy5.5 (clone RPA-T8, Biolegend, UK); CD3 BV605 (Becton Dickinson, UK); CD14 ECD (clone RM052, Beckman Coulter, UK); CD19 ECD (clone J3-119, Beckman Coulter, UK); Live/Dead fixable red stain dye (Life Technologies, UK). Following surface staining, samples were permeabilised and fixed using Fix/Perm solution and buffer (eBioscience, UK). Samples were intracellularly stained with: IL-4 APC (clone 8D4-8, Biolegend, UK); IL-17 Pacific Blue (clone BL168, Biolegend, UK); FOXP3 PE (clone PCH101, eBioscience, UK); IFNy AF700 (clone 4S.B3, Biolegend, UK).

Characterisation of checkpoint marker phenotypes in maternal and decidual samples (N=8) was out carried by staining with: CD3 Amcyan (clone SK7, Becton Dickinson, UK); CD8 ApcCy7 (clone SK1, Biolegend, UK); CCR7 FITC (clone 150503, Becton Dickinson, UK); CD45RA AF700 (clone HI100, Biolegend, UK); CD14 ECD (clone RM052, Beckman Coulter, UK); CD19 ECD (clone J3-119, Beckman Coulter, UK); PD-1 BV421 (clone EH2.2H7, Biolegend, UK); CTLA-4 PeCy5 (clone BNI3, Becton Dickinson, UK); Tim-3 PeCy7 (clone F382E2 Biolegend, UK); Lag-3 (clone FAB2319P, R+D Systems, UK).

Mixed lymphocyte reaction assay

Fresh maternal and decidual mononuclear cells (N=10) were prepared as previously described. T -cell enrichment was performed using Easy Sep T-cell enrichment antibody mix, magnetic beads and Easy Sep Purple Magnet separation (StemCell, UK). The T-cell enriched suspensions were then split between non CD4+ T regulatory depleted and T regulatory depleted samples. Suspensions that were retained for T regulatory depletion were depleted using CD25 Miltenyi Microbeads (Miltenyi, Uk) magnetic MACs micro-column separation. To trace proliferation of cells in the assay, 1ul of Violet cell trace dye (Invitrogen, UK) was added per 10⁶ of cells and incubated at 37 degrees for 20 minutes.

Cord blood mononuclear cells were isolated using method previously described. 5mls of red blood cell lysis buffer was added to each sample and cell suspensions were then centrifuged and re-suspended in 20mls of enriched media. Cord blood lymphocytes were then irradiated for 3000 RADS.

Irradiated cord blood lymphocytes were added at a 2:1 ratio to each maternal and decidual sample in a 96 well round-bottom plate in enriched media. Each sample was run in duplicate and T regulatory cell depleted samples were also run in parallel. Positive control samples were established with the addition 5ul of CD3 dynabeads (Becton Dickinson, UK) instead of cord blood. Negative controls were established in only enriched media. To each sample, 10 Units of IL-2 cytokine was added. Samples were incubated for 4 days. At day three, 100ul of media per well was replaced with fresh media along with 10units of IL-2.

Prior to harvesting on day 4, 1.25ug of monensin was added to each sample and incubated for a further 4 hours. Each sample was harvested and washed with PBS. Surface staining was performed using: CD3 Brilliant violet 605 (clone SK7, Biolegend, UK); CD8 brilliant violet 510 (clone RPAT-8, Biolegend, UK) CD4 PerCP CY5.5 (clone RPAT-4, Biolegend, UK) and Live/Dead fixable red stain dye (Life technologies, UK). Samples were permeabilised and fixed using 100ul of 4%PFA (Sigma, UK) and 10ul of 4% Saponin (Sigma, UK). Intracellular was performed using: IL-10 PE (clone JES3-9D7, eBioscience, UK); IL-4 APC (clone 8D4-8, Biolegend, UK), IL-17 Apc-Cy7 (clone BL168, Biolegend, UK); IFNy AF700 (clone 4S.B3, Biolegend, UK), Granzyme B FITC (clone GB11, Biolegend, UK); Perforin PeCy7 (clone dG9, eBioscience, UK). Samples were washed then re-suspended in MACs before flow cytometry was performed.

HY specific CD8+ T cells in Decidual and Maternal peripheral blood

Mononuclear cells from the decidua basalis and maternal peripheral blood were prepared as previously described (N=9) from pregnancies that resulted in male offspring. Additional HY control samples (N=5) were obtained from pregnancies that resulted in female offspring. Patient samples were selected based on MHC A2 and B7 positivity. Samples were incubated with HY PE dextramer (Immundex, DK), either A2 (FIDSYICQV) or B7 (SPSVDKARAEL) specific, for 20 minutes prior to full flow cytometer staining protocol. Antibodies used were: Live/Dead fixable red stain dye (Life Technologies, UK); CD8 Efluor450 (clone OKT8, eBiosciences, UK); CD3 Amcyan (clone SK7, Becton Dickinson, UK); PD-1 PeCy7 (clone EH12.2H7 Biolegend, UK); CD69 AlexFluor 647 (clone B176236, Biolegend, UK); CD45RA AF700 (clone HI100, Biolegend, UK); CCR7 FITC (clone 150503, Becton Dickinson, UK).

Microarray

12 samples selected for the microarray consisted of six matched maternal and decidual mononuclear cells prepared using previously stated methods. Prior to flow cytometric cell sorting, the following antibodies were applied: CD3 APC (clone UCHT1, Invitrogen, UK); Live/Dead fixable red stain dye (Life Technologies, UK), CD4 PE-Cy7 (clone RPAT4, eBioscience, UK); CD8 PerCp Cy5.5 (clone RPAT8, eBioscience, UK); CD45RA PE (clone HI100, Biolegend, UK); CCR7 FITC (clone 150503, Becton Dickinson, UK). Per maternal and decidual sample, cells were sorted into separate CD4 and CD8 effector memory samples. RNA was immediately isolated and extracted using the Qiagen MINI MICRO PLUS RNA easy kit. RNA samples were immediately stored at -80 degrees. In total, 24 RNA samples were sent to Eurofins Genomics services. Affymetrix Pico labelling kit was used to amplify RNA, and the Affymetrix Human Gene 2.0 ST array was used to carry out the transcriptome analysis. Microarray data accession number: E-MTAB-5517, website URL: www.ebi.ac.uk/arrayexpress

Statistics

Flow cytometry data was analysed using FACS Diva (Becton Dickinson, UK) and statistical analysis was performed using Prism (GraphPad, US). Non Gaussian distribution was applied to all samples, therefore Wilcoxon matched pairs signed rank test was used to identify significance between two sets of data, whilst Friedman’s test with Dunns multiple comparison was used for multiple sets. Microarray data was analysed with the R Limma Package (Bioconductor). Normalisation was performed with the Loess (intra-array) and Aquantile (inter-array) methods. An adjusted p-value (Benjamini and Hochberg's method) of 0.05 and below was taken as significant for differences in gene expression. The empirical Bayesian shrinkage of moderated t-statistics function (eBayes) was used to generate Bayes factors for significantly upregulated or downregulated genes in decidua compared to maternal peripheral blood.

Volcano plots were generated using R and genes annotated based on significant Bayes value (>5). Gene Set Enrichment Analysis was performed using GSEA and MSigDB, (Broad Institute).

Study Approval

Written informed consent was obtained from all women recruited into the study. Matched maternal blood, cord blood and placenta were collected with the approval of Health Research Authority- West Midlands, Edgbaston Research Ethics Committee (RG_14-194).

Results

Highly differentiated effector memory CD4+ and CD8+ T cells dominate the decidual T cell repertoire

In order to investigate the nature of the decidual T cell infiltrate at term (>37 weeks gestation) we obtained decidual tissue collected at the time of elective Cesarean section from 45 women who had an uncomplicated pregnancy. A detailed analysis of the phenotype of lymphocytes obtained from the decidua was undertaken and compared with matched maternal peripheral blood. Flow cytometry was used to determine the T cell memory status according to the pattern of CD45RA and CCR7 co-expression (CD45RA+ CCR7+ _naive, CD45RA- CCR7+ _{central memory}, CD45RA- CCR7- _{effector memory} and CD45RA+ CCR7- _{revertant memory}) (Figure 1, A and B). Strikingly, the dominant population of T cells within the decidua basalis (and parietalis Supplemental Figure 1, A and B) was the effector memory subset which comprised 53% of CD4+ and 51% of CD8+ T cells respectively. These values were markedly increased compared to maternal peripheral blood where the proportions were 30% and 26% respectively (both p= <0.0001) (Figure 1C). In order to investigate the differentiation status of these effector memory (EM) cells in more detail we also examined the pattern of CD27 and CD28 co-expression, which sub-classifies EM T cells within four subsets (CD27+CD28+ _EM1, CD27+CD28- _EM2, CD27-CD28- _EM3 and CD27-CD28+ _EM4) (Supplemental Figure 1, C-D) (27). Decidual effector memory cells were found to be markedly more differentiated than those within maternal blood, with a particularly high proportion of the EM3 subset (CD27- and CD28-). In particular, this subset represented 29% and 33% of decidua-derived CD4+ and CD8+ EM cells respectively compared to 1.8% and 14% in peripheral blood (both p= <0.0001 respectively). Additionally expression of CD57, a marker of late differentiation, was also markedly increased on decidual CD4+ and CD8+ T cells in comparison to maternal peripheral blood (both p=0.0001) (Supplemental Figure 2 A and B).

Figure 1. Decidual CD4+ and CD8+ T cells demonstrate significantly increased cytokine production and a highly differentiated effector memory phenotype.

(A) and (B) Freshly isolated lymphocytes from maternal peripheral blood and decidual mononuclear cells are gated on live CD3+, CD4+ or CD8+ and examined for memory status using CD45RA and CCR7 expression via flowcytometry (n=45). (C) Memory status classified into 4 categories: Naïve (N) CD45RA+ CCR7+; Central Memory (CM) CD45RA- CCR7+; Effector Memory (EM) CD45RA- CCR7-; Effector Memory RA (EMRA) CD45RA+ CCR7-. Effector memory subcategories established using CD28 and CD27 expression: EM1 CD28+ CD27+; EM2 CD28- CD27+; EM3 CD28- CD27-; EM4 CD28+ CD27-. (D) and (E) Peripheral blood and decidual lymphocyte cytokine expression following mitogen stimulation was identified by gating on live CD3+ CD4+ or CD8+, IFNy and IL-4. (F) and (G) Comparison of Maternal PBMC and Decidual total (n=35) and effector memory( n=10) CD4 and CD8 T cell IFNy and IL-4 cytokine expression (Wilcoxson paired samples test, *P=<0.05 **P=<0.01***P=< 0.001).

We then examined the functional capacity of T cells within the two compartments by analysis of intracellular expression of IFN-γ and IL-4 in paired blood and decidual samples (Figure 1, D and E). Expression of both of these cytokines was markedly increased in both the CD4+ and CD8+ decidual T cells compared to peripheral blood. As anticipated, CD4+ T cells showed a lower level of IFN-γ expression and a higher degree of IL-4 production compared to the CD8+ T cells; an average of 25% and 5.4% of CD4+ T cells produced IFN-γ and IL-4 following mitogenic stimulation compared to 60% and 1.2% of CD8+ T cells. However, decidual T cells consistently demonstrated increased expression of both cytokines compared to peripheral blood subsets (p= 0.0004 and <0.0001 for IFN-γ; p= 0.0010 and 0.0006 respectively for IL-4) (Figure 1, F and G). To ensure that these results were not due to the increased proportion of effector memory cells residing in the decidua, CD4 and CD8 effector memory T cells were analysed for IFNγ and IL-4 expression following mitogen stimulation. Both IFNγ and Il-4 were significantly increased in CD8 and CD4 effector memory T cells (p=0.0039 and 0.0078 for IFNγ and p=0.039 and 0.0039 For IL-4 respectively). The proportion of T cells that expressed the chemokine receptor CXCR3 was also measured in non-mitogen stimulated samples (Supplemental Figure 2 C). It was found that CD4 T cells expressing CXCR3 was significantly higher in the decidua, 16.5%, compared to that of maternal PBMCs, 5.6% (p=0.0001).

Increased numbers of T regulatory cells are found within decidua and high level expression of checkpoint proteins is observed on CD4+ and CD8+ effector T-cells

The activation of effector T cells is attenuated by the action of T regulatory cells and the relative balance of these two compartments is important for the maintenance of immune homeostasis. T regulatory cells were identified through a CD3+CD4+CD25^highCD127- and FoxP3+ phenotype (Figure 2A), and consistent with previous reports (28) increased numbers of T regulatory cells were measured in the decidua, 8.1% of CD4+ T cells compared to 3.2% of maternal peripheral blood T cells (p=<0.0001) (Figure 2B).

Figure 2. CD4+ T regulatory Cells and immune checkpoint markers are elevated in the decidua.

(A) CD4+ T regulatory cells were defined by gating on live maternal and decidual CD3+, CD4+ CD127low CD25+ FOXP3+ cells ex vivo (n=35). (B) Comparison of % proportion maternal PBMC and matched decidual CD4 T regulatory Cells (Wilcoxson paired sample test, **P=<0.01). (C) and (D) Immune checkpoint markers PD-1, Tim-3, CTLA-4 and Lag-3 were all measured and their % proportion compared in maternal peripheral blood and matched decidual CD4 and CD8 effector memory T-cells (n=8) (Wilcoxson paired sample test, *P= <0.05, **P= <0.01). Boolean gating analysis was used to analyse dual positivity of these exhaustion markers and their proportion in CD4+ and CD8+ decidual T cells are presented in pie charts.

In addition, we also went on to examine potential mechanisms of intrinsic regulation of effector T cell activation. As such, we determined the expression of inhibitory checkpoint proteins on decidual CD4+ and CD8+ T cells and compared this to maternal peripheral blood (Figure 2, C and D). The expression of PD-1, Tim-3, Cytotoxic T Lymphocyte-Associated Protein 4 (CTLA4) and Lymphocyte Activation Gene 3 (Lag-3) was markedly increased on decidual effector memory T cells as compared to effector memory T cells within peripheral blood. This pattern was seen both for CD4+ and CD8+ T cells and was most marked in the case of PD-1, expressed on 45% and 56% of CD4+ and CD8+ T cells respectively. Tim-3 and CTLA-4 expression was very low on peripheral blood CD4+ T cells, whereas in the decidual expression approached 10%. LAG-3 also significantly increased from 10% to 17% in decidual CD4+ cells. In CD8+ T cells, Tim3 and CTLA-4 were expressed at somewhat higher levels on CD8+ T cells within blood compared to CD4+ cells. However, again the percentage expression of Tim-3, CTLA-4 and LAG-3 significantly increased from 15%, 4%, 8% to 28%, 14%, and 16% respectively on CD8+ decidual cells compared to maternal peripheral blood. Boolean analysis was used to examine the pattern of co-expression of inhibitory proteins. Expression of 2 markers was the most frequent co-expression pattern, and was dominated by PD-1. PD-1 was commonly found co-expressed with either Tim-3 (47%, 37%), CTLA-4 (32%, 21%) or Lag-3 (35%, 41%) in CD4+ and CD8+ T cells respectively (Figure 2, C and D).

Decidual T cells can recognise fetal antigen and are suppressed by T regulatory cells

Having established that decidual T cells contain a high proportion of highly functional effector cells we sought to understand whether this pattern is driven by recognition of fetal antigen. Initially, HLA-peptide multimers containing immunodominant peptides derived from HY and restricted by either HLA-A*0201 or HLA-B*0702 were used to identify HY-specific CD8+ cells in women during pregnancies with male fetuses (Figure 3 and Supplemental Figure 3A). As previously reported (16, 15, 14), HY-specific T cells were identifiable within maternal peripheral blood in gender-mismatched pregnancies, although present at very low frequency, 0.06 % of the total CD8+ T cell population. In contrast, HY-specific CD8 T-cells were markedly increased within decidua increasing 10 fold to represent 0.6% of CD8+ T cells (p= 0.0313) (Figure 3B). Additionally, HY-specific CD8 T cells were not detected in the decidua or peripheral blood of pregnancies that resulted in female offspring, which suggests that any HY specific response in the decidua is due to HY minor histocompatibility antigen present on placental tissue. HY-specific CD8 T cells were predominantly effector memory with very high levels of PD-1 and the activation marker CD69 (52.02% and 65% of total effector memory respectively) compared to maternal peripheral blood. (Figure 3, C and D).

Figure 3. Fetal minorhistocompatability antigen specific CD8+ T cells are present at high levels in the decidua exhibit an activated phenotype and show elevated expression of PD-1.

(A) CD8+ T cell responses to HY-minor histocompatability antigens (restricted on HLA-A2 and HLA-B7) were measured by gating on freshly isolated maternal peripheral blood and matched decidual live CD3+ CD8+ and HY dextramer positive cells, ex vivo, from pregnancies resulting in a male fetus (n=9). As controls an HLA specific isotype tetramer was used per sample and HY specific CD8+ T cells were measured in decidua and peripheral blood of pregnancies resulting in female offspring (HY controls, n=5). (B) HY specific CD8+ T cells are significantly increased in the decidua (Wilcoxon paired samples test, *P=<0.05). Horizontal lines represent median and interquartile range. (C) These HY specific CD8+ decidual T cells (red) are majority effector memory when gating on CD45RA and CCR7 expression. (D) Comparison of CD69 and PD-1 expression by HY-specific CD8 maternal and decidual effector memory T cells (Wilcoxon paired samples test, *P=<0.05).

We assessed the functional response of the decidual T cell population following exposure to matched, irradiated fetal blood cells using a mixed lymphocyte reaction (MLR). The functional response of the decidual T cells was assessed by measurement of proliferation and expression of the cytokines IFN-γ IL-4, IL-10, and the cytotoxicity markers perforin and granzyme (n=10). Decidual CD4+ and CD8+ T cells both demonstrated significantly stronger proliferation than maternal peripheral blood T cells in response to fetal antigen exposure (p= 0.0039 and 0.027 respectively) (Figure 4, B and C). In order to investigate the regulation of this functional response to fetal cells we went on to deplete CD4+ T regulatory cells prior to the MLR assay. CD25 ^high T cell depletion (Supplemental Figure 2 D) led to a substantial increase in both CD4+ and CD8+ T cell proliferation in response to stimulation with cord blood cells (p=0.0020 and 0.0039). Notably proliferation of both CD4+ and CD8+ T cells increased by 1.59 and 1.9 fold in comparison to non T regulatory cell depleted cultures.

Figure 4. Decidual CD4+ and CD8+ T-cells are capable of foetal antigen specific responses.

(A) Example histogram demonstrating differences of proliferation dye dilution between maternal peripheral blood, decidual and decidual T cells depleted for T regulatory cells, following stimulation with irradiated cord blood monocular cells (B and C) Maternal peripheral blood and Decidual T cells were cultured with matched irradiated cord blood mononuclear cells for 4 days and proliferation was measured using violet proliferation dye, n=10 (**P=<0.01 Wilcoxson paired sample test). CD4+ T regulatory cells were depleted in these cultures, and decidual T cell proliferation, granzyme, IL-4 and IL-10 was measured (**P= <0.01 Wilcoxson paired sample test). (D and E) The % proportion of granzyme expressing maternal peripheral blood and decidual CD8+ and CD4+ T cells from the MLR was measured and compared (**P=<0.01 Wilcoxson paired sample test) (F) The % proportion of cytokine Il-4 and Il-10 expression was also measured in maternal peripheral blood and decidual CD4+ T cells from the MLR and compared, n=10, Wilcoxson paired samples test, *P= < 0.05.

Granzyme production in response to fetal antigen exposure during the MLR significantly increased from 2.79% and 17.15% in peripheral blood CD4+ and CD8+ T cells to 8.08% and 32% of decidual CD4+ and CD8+ T cells respectively (Figure 4, D and E). Levels of IL-10 and IL-4 were also markedly increased in decidual CD4+ T cells (3.77% and 11.32% respectively) in comparison to peripheral blood CD4+ T cells (1.27% and 3.42% respectively) following the MLR (Figure 4 F and Supplemental Figure 3). Following T regulatory cell depletion in MLRs, granzyme was significantly increased in decidual CD4+ and CD8+ T cells (p=0.0068 and 0.0010 respectively) and IL-4 was also increased in CD4+ decidual T cells in comparison to maternal peripheral blood T cells (p=0.0039). Overall, these data indicate that decidual CD4+ and CD8+ T cells include T cells with alloreactive specificity against fetal antigen and that these are controlled both intrinsically by high levels of expression of PD-1 and also through the activity of resident regulatory T cells.

Transcriptional analysis of CD4+ and CD8+ effector memory T-cells reveals a strong response in interferon signalling within decidual T cells

In order to comprehensively describe the differences between the decidual and peripheral effector memory T cell population we went on to perform a transcriptional analysis using a microarray of CD4+ and CD8+ effector memory T cells purified from decidual tissue or matched peripheral blood. 26 and 31 genes were significantly upregulated in the decidual CD4+ and CD8+ T cells respectively. In addition, 6 genes were shown to be down-regulated within decidual CD4+ effector T cells (Figure 5, A and B).

Figure 5. Expression profile of Decidual vs Maternal Peripheral Blood CD4+ and CD8+ effector memory T cells.

(A) and (B) RNA isolated from maternal peripheral blood and decidual CD4+ and CD8+ effector memory T cells was analysed using an HTA 2.0 microarray (n=6). Volcano plots were generated based on individual probes log fold change and P value associated with reproducibility of these changes between Maternal peripheral blood and Decidual T cells. Bayesian analysis was used to calculate genes with significantly different expression profiles, represented in red, and appropriately annotated in order of Bayesian value (B≥5). (C) and (D) Genes that had significantly differential expression in deciduda were entered into Database for Annotation, Visualisation and Integrated Discovery (DAVID) and significantly upregulated pathways were generated (Bonferroni corrected p=<0.05). The seven isoforms of CLIC1 are also categorised as well as non coding RNAs.

Eight genes, RSAD2, IFI6, IFI27, IFI44L, IFIH1, EIF2AK2, DDX60 and USP41, were upregulated in both CD4+ and CD8+ from decidua. Interestingly, these are all associated with the cellular response to interferon signalling. RSAD2, also known as viperin, is an interferon-inducible antiviral protein induced by type I and type II interferon. It also facilitates T-cell receptor (TCR)-mediated GATA3 activation and optimal T-helper 2 (Th2) cytokine production (29,30). IFI6, IFI27, IFI44L and IFIH1 are all interferon-inducible genes that are associated with a type 1 interferon signature and EIF2AK2 is a double-stranded RNA-dependent protein kinase R that is amongst the first proteins to become activated following viral infection or stress signalling (31). DDX60, also known as DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), is a cytosolic DNA sensor and RNA helicase that is involved in RLR-mediated type I IFN production after viral infection and mediates viral RNA degradation via the RNA exosome (32). The function of USP41 is unknown but it has high sequence homology with USP18, (USP)-18, a Ubiquitin-specific peptidase which acts as a negative regulator of interferon α/β responses (33). Several differences were observed in the transcriptional profile between CD4+ and CD8+ T cells within decidua including the OAS1, OAS2 and OAS3 genes, which encode members of the 2-5A synthetize family involved in the innate immune response to viral infection and which were upregulated by approximately 2-5 fold in CD8+ decidual T cells. A specific finding of interest within CD4+ T cells was a marked increase in expression of the CLIC1 intracellular chloride channel which has not previously been reported to be expressed in decidual T cells.

Of the 6 significantly downregulated genes in CD4+ effector memory decidual cells, 3 are described as non-coding RNAs. Despite an absence of documented functional relevance in T cell immunology, these non-coding RNAs may have a role in epigenetic regulation of gene expression, and the downregulation of these genes may be distinct in T cell activation in the decidua. Indeed a recent study has suggested several upregulated and downregulated micro RNAS have a potential role in the decidua of patients of recurrent miscarriage(34).

The genes encoding Galectin-1 and Galectin-9 demonstrated increased expression in decidual CD4+ and CD8+ effector memory cells respectively. Galectin-1 expression has been previously observed on decidual NK cells and galectin-9 is also present on T regulatory cells in pregnancy (35,36). As such this particularly high expression by effector T cells within decidua is further evidence that these proteins are important immune-regulatory molecules during pregnancy (37). Further interrogation of the role of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) identified several pathways that were significantly upregulated, most notably ‘immune responses to virus’ and ‘regulation of apoptosis’ (Figure 5, C and D).

We also examined the combined pattern of gene expression within specific immune response and regulatory pathways (Supplemental Table 1). Gene Set Enrichment Analysis demonstrated that gene sets and pathways associated with apoptosis, negative regulation of activation of T cells and IFNγ and IFNα were enriched in decidual T cells. Consistent with our earlier findings, genes encoding checkpoint proteins Lag-3, Tim-3, CTLA-4 and additionally TIGIT were differentially expressed in decidual effector CD8+ and CD4+ T cells, in comparison to peripheral blood, and contributed to the core enrichment of negative regulation of T cell activation gene sets and pathways within decidual T cells. Several molecules such as TRAIL and Casp3 and genes encoding CCR5, Granzyme B, CXCR3, and IFN-γ, were also differentially expressed in decidual effector T cells and contributed to the enrichment of apoptosis and allograft response gene sets within decidual T cells respectively. These findings indicate that CD4+ and CD8+ T cells at the maternal-fetal interface demonstrate a unique transcriptional profile including a dominant response to interferon signalling and activation of inflammatory pathways but also express a wide range of proteins associated with immune regulation.

Discussion

It is established that lymphoid cells play a pivotal role in eutherian reproduction. However, the role of T lymphocytes (which become the major lymphoid subset within the decidua towards term) is much less well characterised but is clearly important given that T cell dysregulation is associated with adverse pregnancy outcomes, especially disorders of malplacentation such as pre-eclampsia and fetal growth restriction. Understanding T cell function at the maternal-fetal interface (within the decidua) is therefore very important for targeted medical advancements in these conditions. In this study we have therefore examined the phenotype, function, transcriptome and specificity of circulating and decidual CD4+ and CD8+ T cells at third trimester.

It is has been previously shown that there is a high concentration of differentiated CD8+ T cells in the human decidua during late pregnancy (18) along with T regulatory cells (28, 38). Here we have analysed both CD8+ and CD4+ subsets, with a comparison with matched maternal blood samples and had the opportunity to combine this with further functional analysis and novel transcriptional analysis of decidual effector T cells. Increased proportions of both CD4+ and CD8+ effector T cells were seen in decidual tissue compared to blood although it is important to note that this may at least partly reflect the observation that naïve T cells are relatively excluded from peripheral tissue. Despite this, several differences were observed in the transcriptional and functional profile of effector cells between blood and tissue. These included increased representation of the CD27-CD28-subset with increased cytolytic properties and shortened telomere length (39) suggesting that these T cells may potentially be activated by local allo-antigen (18). We also demonstrated that decidual effector memory T cells express high levels of IFN-γ and Il-4, an unusual profile that may potentially relate to local effects of progesterone (40). The immunological balance that is maintained at the maternal-fetal interface is regulated not only by cellular processes distinct to maternal immune cells and fetal tissue, but that of maternal hormonal changes at the maternal-fetal interface. Progesterone in particular is known to induce increased levels of IL-4 in maternal CD4+ and CD8+ T cells and limit their cytotoxicity (40, 41).

Given this strong functional capacity, we were particularly interested in the mechanisms that might regulate decidual T cell activation and focussed on mechanisms of extrinsic and intrinsic suppression. Several groups have reported an increase in T regulatory cells within decidua (42) and we were able to confirm this with a 2.5 fold increase in the proportion of T regulatory cells compared to blood. We were further interested to study the expression of inhibitory checkpoint proteins on decidual T cells as these proteins mediate intracellular signalling pathways that limit the magnitude and outcome of T cell responses by negatively regulating T cell activation and function. Indeed, antibodies that block inhibitory receptors are having a transformative impact on the treatment of many forms of cancer (43). We observed that the expression of four of the major checkpoint proteins, PD-1, Tim-3, CTLA-4 and LAG-3, increased substantially on effector memory T cells within decidua. A similar profile has been observed on decidual T cells in the first trimester and indicates that this profile is established early within pregnancy and is maintained throughout gestation (44). PD-L1 expression has been demonstrated on syncytiotrophoblast, and intermediate trophoblastic cells located in the chorion laeve and implantation site, which provides further support for the importance of this inhibitory pathway in reproduction (45).

In this context it is important to consider that the pattern of expression of checkpoint proteins on decidual T cells is comparable to the levels that are expressed on tumour-infiltrating lymphocytes. The use of checkpoint inhibitors for cancer treatment might therefore have a profound impact on the outcome of human pregnancy and should be used with caution in this setting. Some murine models have suggested that interruption of the PD-1:PD-L1 axis can lead to fetal loss but this observation requires further investigation (22,44,46).

Combinatorial staining showed that checkpoint proteins were often expressed together on decidual T cell populations. Again, PD-1 was the dominant partner in each case but 35% of both CD4+ and CD8+ PD-1+ T cells expressed an additional checkpoint protein. This indicates that checkpoint expression is not redundant but the physiological importance of these patterns of expression, and their relevance in relation to checkpoint blockade in oncology, will need further investigation.

Given the evidence of local activation of T cells within decidua it was important to assess if decidual lymphocytes could recognise proteins expressed on fetal tissue. Interestingly, although both CD4+ and CD8+ decidual T cells proliferated in response to cord blood cells this effect was more marked for CD8+ T cells. The profile of paternally-derived antigens presented on cord blood cells will represent only a subset of those expressed on all fetal tissues and as such we believe this assay would have underestimated the total magnitude of the fetal-specific immune response. Nonetheless, importantly, we were able to use it to demonstrate a role for T regulatory cells in the suppression of the decidual T cell response to fetal antigen. T regulatory cell depletion led to a 1.59 and 1.9 fold increase in decidual CD4+ and CD8+ T cell proliferation in response to cord blood and demonstrates the importance of this regulatory population in suppressing maternal response to fetal protein.

In order to investigate the potential importance of inhibitory protein expression on fetal-specific decidual T cells we were able to combine HLA-peptide tetramer staining of HY-specific T cells with assessment of PD-1 expression. This revealed high level expression of PD-1 on HY-specific cells, further implicating this checkpoint protein in an important role in regulating the activity of fetal specific T-cells within the decidual microenvironment. Unfortunately, the low frequency of such cells prevented us from assessing the functional impact of PD-1 inhibition. The overall picture that emerges from these results is that both intrinsic and extrinsic regulation are utilized in control of fetal-specific T cell function within decidua.

Microarray analysis revealed a wide range of genes whose transcriptional activity was differentially expressed in decidual effector memory CD4+ and CD8+ T cells compared to peripheral blood. Significantly, genes associated with interferon signalling receptivity were markedly increased in both CD4+ and CD8+ decidual T cells. Interestingly high levels of interferon-γ have been reported within decidual tissue (47) and this appears to be localised primarily within cells of the monocyte linage (48). Interferon production is driven most strongly by viral infection and the observations raise the question as to whether or not localised viral infection is found within decidual tissue. Indeed, we would suggest that endogenous retroviruses (ERV) are the likely trigger for interferon production within decidua. ERVs represent more than 8% of the human genome (49, 50) and became integrated into the mammalian genome between 0.1 and 40 million years ago via retroviral infection of germ cells (51). Notably, a protein encoded from the env gene of ERVW-1 called Syncytin-1 has an essential role in placentogenesis (52, 53) through formation of the syncytiotrophoblast. Furthermore, syncytium 1 has been shown to be released into the periphery via placental microvesicles that are able to illicit a T cell response (54) and substantial expression of proteins from the endogenous retrovirus HERV-K has been demonstrated in villous and extravillous cytotrophoblast (55). In context to the functional relevance to this type 1 interferon signalling, interferon-α is known to be a significant antiangiogenic factor, resulting in down-regulation of pro-angiogenic factors including VEGF (56). This can result in impairment of vascular remodelling in the case of lupus patients, and it has been suggested that this interferon response can be localised to the decidua resulting in malplacentation and pregnancy complication (57). Together this data may implicate decidual T cells in a role that modulates further vascularisation as pregnancy progresses.

Interestingly our microarray also revealed substantial upregulation of the CLIC1 gene in decidual CD4+ effector T cells. This chloride channel protein has garnered much attention recently within solid-tumors where its expression is associated with metastasis and invasion (58,59,60). CLIC1 is a metamorphic protein that can shift between two or more stable conformations and its expression is increased by hypoxia where it acts to regulate cell function through the ROS-mediated p38 MAPK signalling pathway (59,61). It is known that the placenta develops in a hypoxic environment, and that Hypoxia Inducible Factors (HIFs) play a key role in modulating placental cell function under low oxygen conditions (62).

Our work identifies a new role for CLIC1 in reproductive T cell immunology and more research will be required to establish its physiological function in decidual CD4+ effector memory T cells. Of note CLIC3, which is another member of the CLIC family, has been shown to be important in pre-eclampsia, where increased levels at the syncytiotrophoblast layer result in disruption of membrane potential and downstream apoptosis (63).

Transcription of the LGALS1 gene which encodes Galectin-1 was upregulated in CD4+ T-cells. Galectin-1 is expressed by both endometrial and uterine NK cells during pregnancy (35), and is involved in generation of tolerogenic dendritic cells (64). Intracellular expression of galectin 1 is also observed in activated T cells (65, 66) where it acts to increase sensitivity to the action of extracellular Galectin-1, in which it activates downstream apoptosis (37). In contrast, decidual CD8+ T cells upregulate galectin-9, a ligand of Tim-3 which is involved in both chemo-attraction and apoptosis (67) as well as inhibition of chronic inflammation (68). Expression of Galectin-9 by T regulatory cells promotes stability of a immunosuppressive phenotype (69) and its expression by decidual CD8+ T cells may therefore increase effector cell regulation following interaction with Tim-3 (37).

The observation that highly differentiated fetal-reactive effector T cells are found within the decidua in healthy pregnancy raises questions regarding their potential role. HY dextramer staining was consistent with previous findings using MHC-peptide multimers when identifying human T cell responses to low affinity minor-histocompatibility antigens (14,15). These maternal immune responses are likely to have been primed following systemic transfer of fetal cells and placental debris into the maternal circulation, which may involve either local or systemic presentation within lymphoid tissue. It should be noted that the HLA-peptide multimers used in the study included the HLA alleles of HLA-A2 and HLA-B7 and these proteins are not expressed on fetal syncytiotrophoblast or extravillous trophoblast (EVT). As such it remains possible that anatomical limitations may serve to largely limit the potential interaction between fetal-specific T cells and fetal tissue. In this regard it will be important in future studies to investigate the presence and function of fetal-specific cells restricted by HLA-C and HLA-G, which are the only polymorphic HLA molecule expressed on EVT.

Indeed, the finding that human decidua contains high numbers of T cells, at least some of which respond to fetal antigens, is provocative in relation to the potential role of this response in disorders of pregnancy. Pre-eclampsia is associated with poor trophoblast invasion and impaired remodelling of maternal arteries, and strong evidence for a role for suboptimal NK activation has been established (70). The potential contribution of alloreactive T cells to this disorder has been more difficult to determine (71, 13,72) and this may reflect the observation that the initial features of pre-eclampsia are established early in gestation, prior to the accumulation of a significant T cell infiltrate (72). Despite this, T cell-mediated immunopathology may be implicated in specific subtypes of pre-eclampsia and transcriptional analysis of placental tissue has recently identified a subset of disease associated with immune infiltration and inflammation (73). Moreover, it is now recognised that villitis of unknown aetiology (VUE) may result from recognition of fetal tissue by the humoral and cellular components of the maternal adaptive immune system (74). The clinical expression of VUE includes fetal growth restriction, preterm birth and recurrent pregnancy loss and the histology is characterized by a heavy maternal inflammatory cell infiltrate of T cells and macrophages with a predominant CD8+ profile (75,76,77). This is associated with focal inflammation of fetal villi and it is noteworthy that this profile is present to a modest extent in many normal pregnancies. The evolutionary balance of fetal-specific T cell responses may be delicately poised to permit successful reproduction in the great majority of cases, whilst avoiding intensive local or systemic maternal immune suppression.

Our study reinforces the emerging concept that T cells play an increasingly important role at the maternal-fetal interface and that fetal-specific recognition is carefully modulated by multiple checkpoint proteins and T regulatory cells. Improved understanding of immune homeostasis within this unique microenvironment is likely to have substantial implications for many areas of clinical immunology.