Pregnancy-associated diseases are characterized by the composition of the systemic regulatory T cell (T_reg) pool with distinct subsets of T_regs

Abstract

Dysregulations concerning the composition and function of regulatory T cells (T_regs) are assumed to be involved in the pathophysiology of complicated pregnancies. We used six-colour flow cytometric analysis to demonstrate that the total CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3)⁺ T_reg cell pool contains four distinct T_reg subsets: DR^high+CD45RA^-, DR^low+CD45RA^-, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs. During the normal course of pregnancy, the most prominent changes in the composition of the total T_reg cell pool were observed between the 10th and 20th weeks of gestation, with a clear decrease in the percentage of DR^high+CD45RA^- and DR^low+CD45RA^- T_regs and a clear increase in the percentage of naive DR^-CD45RA⁺ T_regs. After that time, the composition of the total T_reg cell pool did not change significantly. Its suppressive activity remained stable during normally progressing pregnancy, but decreased significantly at term. Compared to healthy pregnancies the composition of the total T_reg cell pool changed in the way that its percentage of naive DR^-CD45RA⁺ T_regs was reduced significantly in the presence of pre-eclampsia and in the presence of preterm labour necessitating preterm delivery (PL). Interestingly, its percentage of DR^high+CD45RA^- and DR^low+CD45RA^- T_regs was increased significantly in pregnancies affected by pre-eclampsia, while PL was accompanied by a significantly increased percentage of DR^-CD45RA^- and DR^low+CD45RA^- T_regs. The suppressive activity of the total T_reg cell pool was diminished in both patient collectives. Hence, our findings propose that pre-eclampsia and PL are characterized by homeostatic changes in the composition of the total T_reg pool with distinct T_reg subsets that were accompanied by a significant decrease of its suppressive activity.

Introduction

For decades, immunologists have been engaged in the investigation of the cellular mechanisms which prevent the rejection of the semi-allogeneic fetus during the normal course of pregnancy. It is known that special tolerance-inducing mechanisms, such as the expression of Fas-ligand (FasL), human leucocyte antigen-G (HLA-G) and indolamine-2,3-dioxygenase (IDO) in the syncytiotrophoblast act locally at the feto–maternal interface [1–3]. However, a bi-directional feto–maternal trafficking of cells during pregnancy is now well established [4], and it was shown that the maternal immune system is increasingly stimulated with ongoing pregnancy [5]. Therefore, further systemic changes are necessary to antagonize maternal immune reactions which could cause the rejection of the fetus, expressing paternal alloantigens. Currently, regulatory T cells (T_regs) are assumed primarily to be involved in the maintenance of tolerance during pregnancy. It has been reported that murine pregnancy is associated with elevated T_reg numbers in the circulation [6], and that these T_regs prevent the immunological rejection of the fetus both in mice and humans [7,8]. However, in humans, changes of peripheral T_reg cell counts during the normal course of pregnancy still remain a controversial issue. Earlier studies document increased T_reg cell counts in pregnant women compared to non-pregnant women [9], while more recently it was postulated that T_reg cell counts decreased during the normal course of pregnancy [10–12]. Obviously, the number of T_regs depends strongly on the gating strategy by which the T_regs were characterized after staining cells for flow cytometric analysis and on the moment at which the T_reg cell numbers were determined during the course of pregnancy. Similarly, reports evaluating functionality of T_regs are also inconsistent. A decrease of their suppressive activity only at the end of normal pregnancy is reported by our group [13], while most others did not detect any differences in the suppressive activity between non-pregnant and pregnant women [11,14].

As T_reg cells were shown to guarantee the maintenance of feto–maternal tolerance during the normal course of pregnancy, it is assumed that dysregulations concerning their number and function are involved in the pathogenesis of complications, such as gestation-associated hypertensive diseases and preterm intrauterine activation. Currently, decreased peripheral T_reg cell numbers are reported for pregnant women with pre-eclampsia [14–16], deficiencies of their functional activity were ascertained by our group, especially for patients with pre-eclampsia [16], and for patients with preterm labour necessitating preterm delivery [13]. Thereby, we demonstrated that magnetically selected CD4⁺CD127^low+/−CD25⁺ T_reg cells contain a distinct T_reg cell subset expressing very high levels of forkhead box transcription factor protein 3 (FoxP3) and human leucocyte antigen-DR (HLA-DR). HLA-DR expression of these FoxP3⁺DR⁺ T_reg cells was reduced significantly, not only in preterm-labouring women, but also in kidney transplanted patients with acute rejection. The suppressive activity of the whole T_reg pool declined in parallel to the number of FoxP3⁺DR^high+ T_reg cells, disclosing the important role of this T_reg subpopulation for the maintenance of tolerance [13]. Recently, such non-proliferating DR⁺ T_regs were shown to express higher levels of FoxP3 and induced a more vigorous rapid T cell suppression than the T_regs that lack HLA-DR expression [17]. In particular, these highly suppressive DR⁺ T_regs were found to become inactivated by highly activated CD4⁺ responder T cells which produced high amounts of Granzyme B, and it was shown that these DR⁺ T_regs were much more prone to undergo Granzyme B-induced cell death than the DR^- T_regs[18]. Although the majority of DR⁺ and DR^- T_reg cells in adults represent CD45RO⁺ memory T_reg cells, a considerable percentage of naive CD45RA⁺ T_regs can also be detected in the circulation of adults. However, this percentage decreases with increasing age [19]. Recently, an impaired functional activity of such naive CD45RA⁺ T_regs was ascertained for patients with multiple sclerosis (MS) [20], and there is a growing body of evidence that the composition of the total T_reg cell pool with different T_reg cell subsets potentially influences its suppressive activity.

In this study, we used six-colour flow cytometric analysis to demonstrate that the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool consists of four distinct T_reg cell subsets: DR^high+CD45RA^-, DR^low+CD45RA^-, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs. Compared to non-pregnant women, the composition of the total T_reg pool changed in the way that its percentage of naive DR^-CD45RA⁺ T_regs increased, while that of DR^high+CD45RA^- and DR^low+CD45RA^- T_regs decreased during the normal course of pregnancy. In the presence of complications, such as pre-eclampsia or preterm labour necessitating preterm delivery (PL), the composition of the total T_reg pool was strongly modified and its suppressive activity was strongly diminished. Therefore, it could be assumed that the occurrence of certain gestational diseases is associated with characteristic homeostatic changes in the composition of the total T_reg cell pool and that such changes may have a potential effect on its suppressive activity.

Methods

Patient collectives and healthy volunteers

Blood samples were obtained from 31 non-pregnant fertile female volunteers (group A), 135 healthy pregnant women during the normal course of pregnancy [group 1 (11–14 weeks' gestation, group 2 (15–23 weeks' gestation), group 3 (24–36 weeks' gestation), group 4 (37 42 weeks' gestation)], 27 pregnant women affected by pre-eclampsia (group 5, 27–39 weeks' gestation), 15 pregnant women affected by haemolysis-elevated liver enzyme levels-low platelet count syndrome (HELLP) syndrome (group 6, 24–39 weeks' gestation) and 54 pregnant women affected by preterm intrauterine activation (23–36 weeks' gestation) which was diagnosed in the presence of cervical insufficiency or preterm labour. All these women received tocolytic and corticoid treatment until fetal lung maturity (34 weeks' gestation) was achieved. These patients were grouped as cervical insufficiency (CI) in cases of cervical shortening of less than 20 mm before 36 completed weeks' gestation in the absence of labour leading to preterm delivery (group 7, n = 30, 24–35 weeks' gestation). The diagnosis of preterm labour necessitating preterm delivery (PL) was made in the case of the occurrence of spontaneous preterm labour (before 36 completed weeks' gestation) which was resistant to tocolytic treatment and which led to irresistible preterm delivery (group 8, n = 24, 23–36 weeks' gestation). Blood samples from healthy pregnancies (groups 1–4) were collected from women who had routine ultrasonography to exclude fetal malformations (groups 1–3) and from women delivering spontaneously or by elective caesarean section at term (group 4). Blood samples from affected pregnancies were collected during their hospitalization due to diagnosis of pre-eclampsia (group 5), HELLP syndrome (group 6), CI (group 7) or PL (group 8). Pre-eclampsia was diagnosed as blood pressure higher than 140/90 mm Hg on two separate occasions, 6 h apart, along with significant proteinuria (>300 mg/l in a 24-h collection or a dipstick reading of >2+ on a voided random urine sample in the absence of urinary tract infection) in previously normotensive women. The diagnosis of HELLP syndrome was made on the basis of haemolysis, elevated liver enzyme levels and thrombocytopenia. The laboratory parameters of these patients were thrombocyte count < 150·000 cell/µl and aspartate aminotransferase and alanine aminotransferase > 30 U/l. All patients in this group had significant proteinuria and showed characteristic clinical symptoms such as left-sided epigastric pain, flickering in front of the eyes and hyperreflexia. The study was approved by the Regional Ethics Committee. All women were fully informed of the aim of the study and informed consent was obtained from all participants.

Both the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells of total CD4⁺ T cells and the percentage of DR^high+CD45RA^- T_regs, DR^low+CD45RA^- T_regs, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs within the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool were determined by six-colour flow cytometric analysis for all participants. In order to control whether the results obtained on the percentages of the different T_reg subsets within the total T_reg cell pool correspond to their absolute numbers within the CD4⁺ T cell pool, their percentages were additionally calculated relative to the total CD4⁺ T cell pool. For a total number of six healthy volunteers, 33 healthy pregnant women, 15 pregnant women affected by gestation-associated hypertensive diseases (pre-eclampsia and HELLP syndrome) and 18 pregnant women affected by preterm intrauterine activation (CI and PL) suppression assays were performed in order to test the suppressive capacity of the magnetically selected total CD4⁺CD127^low+/−CD25⁺ T_reg cell pool.

Fluorescence-activated cell sorter (FACS) staining

Venous blood samples (10 ml) from all participants were collected into ethylenediamine tetraacetic acid (EDTA)-containing tubes. Whole peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Amersham Bioscience, Freiburg, Germany) gradient centrifugation and analysed by six-colour flow cytometric analysis. Briefly, PBMCs (4 × 10⁶ cells) were surface-stained with peridinin chlorophyll (PerCP)-conjugated anti-CD4 (BD Bioscience, Heidelberg, Germany), phycoerythrin (PE)-conjugated anti-CD127 (eBioscience, Frankfurt, Germany), allophycocyananin-cyanin 7 (APC-Cy7)-conjugated anti-CD25, PE-Cy7-conjugated anti-HLA-DR (BD Bioscience) and APC-conjugated anti-CD45RA (BD Bioscience) mouse monoclonal antibodies. Subsequently, intracellular staining for the detection of FoxP3 was performed using a fluorescein isothiocyanate (FITC)-labelled anti-human FoxP3 staining set (clone PCH101, eBioscience) according to the manufacturer's instructions. Both the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells and the percentages of the four T_reg subsets (DR^high+CD45RA^- T_regs, DR^low+CD45RA^- T_regs, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs) within the total T_reg cell pool were determined for all participants. For all women, the percentages of these T_reg subsets were additionally calculated relative to the total CD4⁺ T cell pool. Negative control samples were incubated with isotype-matched antibodies. Dead cells were excluded by forward- and side-scatter characteristics. Cells were analysed by a FACS Canto cytometer (BD Bioscience). Statistical analysis was based on at least 100 000 gated CD4⁺ T cells.

Positive selection and staining of CD4⁺CD127^low+/−CD25⁺ T_reg cells

Whole peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood (50 ml) drawn in EDTA tubes by Ficoll-Hypaque (Amersham Bioscience) gradient centrifugation. CD4⁺CD127^low+/−CD25⁺ T_reg cells were purified using the CD4⁺CD127^low+/−CD25⁺ Regulatory T cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. First, CD4⁺CD127^low+/− T cells were isolated by magnetic depletion of non-CD4⁺CD127^high+ T cells. In a second step, the CD4⁺CD127^low+/−CD25⁺ T_reg cells were isolated by positive selection over two consecutive columns. The CD4⁺CD127^low+/−CD25^- T cells were obtained in the flow-through fraction and used as responder T cells (T_resp). The CD4⁺CD127^low+/−CD25⁺ T_reg cells were subsequently retrieved from the columns. The purified CD4⁺CD127^low+/−CD25⁺ T_reg cell fraction was analysed using four-colour flow cytometry. Briefly, 1 × 10⁵ cells were stained with 10 µl PerCP-conjugated-anti-CD4, PE-conjugated anti-CD25, FITC-conjugated FoxP3 and biotin-conjugated anti-CD127 mouse monoclonal antibodies. Positive staining for CD127 was detected using APC-conjugated streptavidin molecules. On average, 85% of the isolated CD4⁺CD127^low+/−CD25⁺ T_reg cells were shown to be within the CD4⁺CD127^low+/−CD25⁺Foxp3⁺ T_reg cell population.

Co-culture suppression assay

Whole peripheral mononuclear cells (PBMCs) were isolated from peripheral blood drawn in EDTA tubes by Ficoll-Hypaque (Amersham Bioscience) gradient centrifugation. CD4⁺CD127^low+/−CD25⁺ T_reg cells were purified using the CD4⁺CD127^low+/−CD25⁺ Regulatory T cell Isolation Kit II (Miltenyi Biotec) described above. In all assays, 2 × 10⁴ T_resp were co-cultured with the purified CD4⁺CD127^low+/−CD25⁺ T_reg cells at ratios 1:1–1:64 in 96-well v-bottomed plates. Suppression assays were performed in a final volume of 100 µl/well of X-VIVO15 medium (Lonza, Verviers, Belgium). For T cell stimulation, the medium was supplemented with 1 µg/ml anti-CD3 and 2 µg/ml anti-CD28 antibodies (eBioscience). As controls, CD4⁺CD127^low+/−CD25⁺ T_reg cells and T_resp cells alone were cultured both with and without any stimulus. Cells were incubated at 37°C and 5% of CO₂. After 4 days 1 µCi [³H]-thymidine was added to the cultures and cells were incubated further for 16 h. Cells were then harvested and [³H] incorporation was measured by scintillation counting. For each donor, the co-culture suppression assay was repeated six times and exhibited <10% standard error of the mean (s.e.m.). For each patient group, the co-culture suppression assays were performed with blood samples obtained from at least six different donors. In order to compare the suppressive capacity of the isolated CD4⁺CD127^low+/−CD25⁺ T_regs between the different patient groups, we calculated the maximum suppressive activity (ratio of T_reg cells to T_resp 1:1). In addition, we determined the suppressive activity of the isolated T_reg cells with gradient ratios of T_reg cells to T_resp cells (ratio of T_reg cells to T_resp cells 1:1–1:64) and identified the ratio with which a minimum suppression of at least 15% could be achieved (titre T_reg/T_resp). A minimum suppression value of 15% was determined, because this value was the lowest which was able to guarantee a significant reduction of the suppressive activity.

Sorting and functional testing of the DR^high+CD45RA^- T_reg cell subset

Venous blood samples (100 ml) from three different healthy, non-pregnant volunteers were collected into EDTA-containing tubes. Whole peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Amersham Bioscience) gradient centrifugation. For fluorescence activated cell sorting of the DR^high+CD45RA^- T_reg cell subset, CD4⁺CD127^low+/−CD25⁺ T_reg cells were purified using the CD4⁺CD127^low+/−CD25⁺ Regulatory T cell Isolation Kit II (Miltenyi Biotec), as described above. Respectively, 5 × 10⁵ cells of the isolated CD4⁺CD127^low+/−CD25⁺ T_reg cells were stained with 20 µl FITC-conjugated anti-CD4 (BD Bioscience), 10 µl PE-conjugated anti-CD25 (BD Bioscience) and 20 µl PE-Cy7-conjugated anti-HLA-DR (BD Bioscience) mouse monoclonal antibodies. Contaminating non-CD4⁺ T cells were excluded, while the remaining cells were sorted using a FACS-VantageSE-Sorter (BD Bioscience). Thereby, the CD4⁺CD127^low+/−CD25⁺ T_reg cells were divided into a T_reg population consisting exclusively of DR^high+CD45RA^- T_reg cells and a T_reg population consisting of the remaining DR^low+CD45RA^- T_regs, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs. Subsequently, the suppressive activity of both T_reg populations was analysed using the above-described suppression assay.

Statistical analysis

Statistical comparison of the percentages of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells within CD4⁺ T cells and of the percentages of the different T_reg subsets (DR^low+CD45RA^- T_regs, DR^high+CD45RA^- T_regs, DR^-CD45RA^- T_regs and naive DR^-CD45RA⁺ T_regs) within both the total CD4⁺ T cell pool and the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool, between the different patient populations, was performed using the non-parametric Kruskal–Wallis H-test, which is used for simultaneous comparison of more than two sample populations. Each H-test was followed by a Dunn test. Comparison of the suppressive activity of purified CD4⁺CD127^low+/−CD25⁺ T_reg cells was also performed using the Kruskal–Wallis test. P < 0·05 was considered significant.

Results

The suppressive activity, but not the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells within the total CD4⁺ T cell pool is reduced significantly in both patients affected by pre-eclampsia and patients affected by preterm labour leading to preterm delivery

Both the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells within total CD4⁺ T cells and their composition with four distinct T_reg cell subsets were determined in the circulation of non-pregnant female volunteers (group A), healthy pregnant women during the normal course of pregnancy (groups 1–4) and in pregnant women affected by pre-eclampsia (group 5), HELLP syndrome (group 6), CI (group 7) or PL (group 8). Peripheral blood mononuclear cells (PBMCs) obtained from each participant were stained with anti-CD4, anti-CD127, anti-CD25, anti-FoxP3, anti-HLA-DR and anti-CD45RA monoclonal antibodies and analysed by six-colour flow cytometric analysis. Figure 1 shows the gating strategy for these measurements. First, CD4⁺ T cells (Fig. 1a, P1) were analysed for their simultaneous expression of CD25, CD127 and FoxP3 (Fig. 1b, P2; c, P3). At first, the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells (Fig. 1c, P3) within the total CD4⁺ T cell pool and their composition with four distinct subsets [DR^high+CD45RA^- T_regs (P4), DR^low+CD45RA^- T_regs (P5), DR^-CD45RA^- T_regs (P6), and naive DR^-CD45RA⁺ T_regs (P7)] were determined for each participant (Fig. 1d). Figure 1a–d shows the gating strategy of one representative experiment for healthy pregnancies; Fig. 1e–h shows the gating strategy of one representative experiment performed with patients affected by HELLP syndrome, pre-eclampsia, CI and PL, respectively.

Fig. 1.

Gating strategy for six-colour flow cytometric detection of the total CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3⁺) regulatory T cell (T_reg) cell pool and its percentage of naive DR^-CD45RA⁺ T_regs, DR^-CD45RA^- T_regs, DR^low+CD45RA^- T_regs and DR^high+CD45RA^- T_regs. (a) First, CD4⁺ T cells (P1) were gated by fluorescence intensity of CD4 versus side light-scatter (SSC). (b) CD4⁺CD127^low+/−CD25⁺ T_reg cells were gated by fluorescence intensity of CD25 versus CD127 (P2). (c) CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells were gated by excluding cells without FoxP3 expression (P3). (d) The percentage of the DR^high+CD45RA^- (P4), the DR^low+CD45RA^- (P5), the DR^-CD45RA^- (P6) and the naive DR^-CD45RA⁺ T_reg subset (P7) was estimated by analysing CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells (P3) for their expression of human leucocyte antigen D-related (HLA-DR) and CD45RA. (a–d) Shows a representative experiment for healthy pregnancies, (e–h) show representative experiments for patients with HELLP syndrome (e), pre-eclampsia (f), CI (g) and PL (h). CI: cervical insufficiency; PL: preterm labour necessitating preterm delivery; HELLP syndrome: haemolysis-elevated liver enzyme level-low platelet count syndrome.

In analogy to our previous studies, when using three- or four-colour flow cytometric analysis for the detection of CD4⁺CD25⁺FoxP3⁺ T_reg cells [16] or CD4⁺CD127^low+/−CD25⁺ T_reg cells [13], we observed a decreasing percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_regs within the total CD4⁺ T cell pool during the normal course of pregnancy. Compared to healthy pregnant women in the first-trimester (group 1), the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells was reduced significantly in term pregnant women (group 4) (Fig. 2a,b). However, in the present study, we did not detect a significantly decreased percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells of CD4⁺ T cells in patients with gestation-associated hypertensive diseases (pre-eclampsia, HELLP syndrome) or preterm intra-uterine activation (CI, PL) compared to healthy third-trimester pregnancies (groups 3 and 4) (Fig. 2a,b).

Fig. 2.

Detection of CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3⁺) T_reg cells and their suppressive activity in healthy and complicated pregnancies. The percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_regs of total CD4⁺ T cells was estimated in pregnancies affected by pre-eclampsia () or HELLP syndrome () (a), and in pregnancies affected by CI () or PL () (b), in comparison to healthy pregnancies (♦). CD4⁺CD127^low+/−CD25⁺ T_reg cells were isolated by the magnetic affinity cell sorting (MACS) technique and their suppressive activity was examined using suppression assays (c) (see Methods). The figure shows the maximum suppressive activity (T_reg/T_resp = 1/1) (d,e) and the ratio of T_reg/T_resp (titre) up to which the purified T_reg cells could be diluted to achieve a minimum suppressive activity of at least 15% (f,g). The diagrams represent the individual data and the regression line obtained for healthy non-pregnant volunteers (group a; n = 6), healthy pregnancies during the normal course of pregnancy (groups 1–4; n = 36), pregnancies affected by pre-eclampsia (group 5, n = 10) or HELLP syndrome (group 6; n = 9) (d,f) and pregnancies affected by CI (group 7, n = 10) or PL (group 8; n = 10) (e,g). Compared to healthy third-trimester women (groups 3 and 4), the suppressive activity of the isolated CD4⁺CD127^low+/−CD25⁺ T_regs from women with pre-eclampsia (group 5) and from women with PL (group 8) was reduced significantly, concerning both the maximum suppressive activity (P < 0·01) and the ratio of T_reg/T_resp up to which a minimum suppressive activity of 15% could be achieved (P < 0·001). CI: cervical insufficiency; PL: preterm labour necessitating preterm delivery; HELLP syndrome: haemolysis-elevated liver enzyme level-low platelet count syndrome.

In order to examine whether the suppressive activity of the total CD4⁺CD127^low+/−CD25⁺ T_reg cell pool from affected pregnancies was impaired compared to healthy pregnancies, we used a co-culture suppression assay [13]. The suppression assays were performed using magnetic affinity cell sorting (MACS)-sorted CD4⁺CD127^low+/−CD25⁺ T_reg cells obtained from six non-pregnant volunteers (group A), 36 healthy pregnant women during the normal course of pregnancy (groups 1–4) and from 10 pregnant women affected by pre-eclampsia (group 5), HELLP syndrome (group 6), CI (group 7) and PL (group 8), respectively. Figure 2c shows the results of one representative experiment for healthy third-trimester women. Figure 2d–g and Table 2 show the results of these suppression assays for affected pregnancies in comparison to healthy pregnancies. The maximum suppressive capacity (T_reg/T_resp = 1/1) of isolated CD4⁺CD127^low+/−CD25⁺ T_regs obtained from healthy first- and second-trimester women was in the same range as that of non-pregnant women. It remained relatively stable during the normal course of pregnancy (groups 1, 2 and 3). However, after 30 weeks of gestation it decreased continuously and reached significantly reduced levels (P < 0·05) at the end of pregnancy near term (group 4, Fig. 2d,e). In addition, we determined the suppressive activity of the isolated T_reg cells using gradient ratios of T_reg cells to responder cells (T_reg/T_resp 1:1–1:64) and identified the ratio with which a suppression of at least 15% could be achieved (titre T_reg/T_resp). During the normal course of pregnancy, this ratio was in the same range as that of non-pregnant women, but it was also decreased significantly (P < 0·05) at the end of pregnancy near term (group 4) (Fig. 2f,g). Hence, the suppressive activity of the T_reg cells ceased considerably near term.

Table 2.

Percentage of the CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3⁺) regulatory T cell (T_reg) cell pool within CD4⁺ T cells, its suppressive activity and its composition with distinct T_reg subsets in healthy and affected pregnancies

	Healthy pregnancy groups 3 and 4	Pre-eclampsia group 5	HELLP syndrome group 6	CI group 7	PL group 8
CD4+ T cells (%)	29·7 (13·1–48·9)	28·9 (7·1–42·6)	29·7 (12·5–40·2)	33·7 (9·7–52·1)	22·0 ↓ (9·0–46·7)
Treg cells (%)	4·4 (2·1–8·7)	4·6 (2·1–6·7)	4·3 (3·0–6·5)	4·7 (2·1–8·9)	4·5 (1·8–7·3)
Suppressive activity
Max. suppr. activity (%) (Treg/Tresp 1/1)	59·9 (35·8–93·8)	37·3 ↓ (7·9–59·0)	82·2 (62·9–89·9)	50·9 (25·0–90·9)	28·2 ↓ (0–68·9)
Titre of Treg (>15% suppr. activity)	1:16 (1:2–1:32	1:1 ↓ (1:1–1:4)	1:16 (1:4–1:32)	1:4 (1:1–1:16)	1:1 ↓ (1:1–1:8)
Subsets
of the Treg cell pool (%)
DR-CD45RA Tregs	38·6 (19·1–68·9)	27·9 ↓ (8·1–48·6)	33·6 (15·0–57·6)	35·4 (11·4–60·9)	26·5 ↓ (7·1–39·8)
DR-CD45RA- Tregs	30·4 (13·9–48·2)	30·5 (18·8–46·8)	25·4 (18·7–53·3)	33·4 (16·1–46·5)	36·1 ↑ (28·0–59·6)
DRlow+CD45RA- Tregs	23·6 (12·4–39·6)	27·1 ↑ (21·8–43·0)	26·4 (15·5–40·0)	24·8 (12·8–36·6)	28·2 ↑ (20·1–55·5)
DRhigh+CD45RA- Tregs	4·8 (1·4–11·1)	6·1 ↑ (0·1–27·7)	6·7 ↑ (3·7–18·0)	4·1 (1·0–9·8)	5·3 (0·9–10·3)
of the CD4+ T cell pool (%)
DR-CD45RA+ Tregs	2·4 (1·0–5·4)	1·5 ↓ (0·5–4·0)	1·7 (1·0–2·8)	2·0 (1·1–4·4)	1·6 ↓ (0·3–2·8)
DR-CD45RA- Tregs	1·2 (0·5–2·5)	1·2 (0·6–2·5)	0·9 (0·5–1·9)	1·4 (0·6–2·7)	1·5 ↑ (0·6–2·5)
DRlow+CD45RA- Tregs	1·1 (0·6–2·3)	1·6 ↑ (0·8–2·5)	1·2 (0·7–3·0)	1·2 (0·5–3·9)	1·7 ↑ (0·7–2·9)
DRhigh+CD45RA- Tregs	0·2 (0–0·6)	0·3 ↑ (0·1–1·2)	0·3 ↑ (0·1–0·9)	0·1 (0–0·5)	0·2 (0–0·5)

↑↓Significant differences were obtained compared to healthy pregnancies; CI: cervical insufficiency; HELLP: haemolysis-elevated liver enzyme levels-low platelet count syndrome; PL: preterm labour necessitating preterm delivery.

Tables 1 and 2 summarize the data regarding the maximum suppressive activity (T_reg/T_resp 1:1) and the ratio of T_reg/T_resp cells that led to a minimum suppressive activity of less than 15%. Compared to healthy third-trimester women (groups 3 and 4, 24–42 weeks' gestation), the suppressive activity of the isolated CD4⁺CD127^low+/−CD25⁺ T_regs from women with pre-eclampsia (group 5) and from women with PL (group 8) was reduced strongly concerning the maximum suppressive activity (P < 0·01) and concerning the ratio (titre T_reg/T_resp) with which a minimum suppressive activity of 15% could be achieved (P < 0·001) (Fig. 2d–g and Table 2). Surprisingly, the suppressive activity of T_reg cells from women with HELLP syndrome (group 6) was in the normal range, similar to that of T_reg cells from healthy third-trimester women (groups 3 and 4). The suppressive activity of T_reg cells from women with CI (group 7) was slightly reduced. However, a significant difference compared to healthy third-trimester women (group 3) was not achieved (Fig. 2d–g and Table 2).

Table 1.

Percentage of the CD4⁺CD127^low+/−CD25⁺FoxP3⁺ regulatory T cell (T_reg) cell pool within CD4⁺ T cells, its suppressive activity and its composition with distinct Treg subsets in non-pregnant women and healthy pregnant women during the normal course of pregnancy

	Group A non-pregnant	Group 1 11–14 weeks	Group 2 15–23 weeks	Group 3 24–36 weeks	Group 4 37–42 weeks
CD4+ T cells (%)	37·4 (24·7–50·8)	29·3 ↓ (21·2–46·8)	31·9 ↓ (16·7–50·4)	28·8 ↓ (16·7–48·9)	32·8 ↓ (13·1–46·2)
Treg cells (%)	4·7 (3·2–7·4)	5·1 (3·2–7·7)	4·5 (2·0–6·8)	4·9 (2·1–8·7)	4·3 (2·8–7·0)
Suppressive activity
Max. suppr. activity (%) (Treg/Tresp 1/1)	84·5 (69·7–94·1)	84·8 (70·1–91·0)	71·3 (66·1–87·6)	74·1 (41·5–93·8)	55·2 ↓ (35·8–85·0)
Titre of Treg (>15% suppr. activity)	1:16 (1:16–1:32)	1:32 (1:16–1:64)	1:16 (1:8–1:64)	1:16 (1:2–1:32)	1:8 ↓ (1:2–1:32)
Subsets
of the Treg cell pool (%)
DR-CD45RA+ Tregs	28·5 (9·8–51·5)	31·9 (17·9–47·9)	37·2 (21·0–55·9)	36·2 (21·7–55·7)	39·1 ↑ (19·1–68·9)
DR-CD45RA- Tregs	30·7 (19·8–43·9)	32·7 (21·4–42·3)	31·6 (21·5–40·2)	30·6 (18·7–48·2)	30·2 (13·9–41·0)
DRlow+CD45RA- Tregs	28·8 (20·6–44·4)	29·3 (15·7–42·9)	24·1 (11·6–36·9)	23·8 (14·3–39·6)	23·5 ↓ (12·4–31·4)
DRhigh+CD45RA- Tregs	8·3 (4·2–20·0)	8·0 (2·7–14·8)	5·0 (1·9–8·8)	5·4 (1·6–9·0)	4·4 ↓ (1·4–11·1)
of the CD4+ T cell pool (%):
DR-CD45RA+ Tregs	1·9 (0·8–3·5)	2·0 (1·0–3·8)	2·3 (1·0–3·9)	2·3 (1·0–5·4)	2·4 ↑ (1·3–4·4)
DR-CD45RA- Tregs	1·5 (0·7–2·4)	1·6 (0·9–2·8)	1·3 (0·9–2·1)	1·2 (0·5–2·5)	1·1 (0·1–2·1)
DRlow+CD45RA- Tregs	1·5 (0·9–3·6)	1·5 (0·7–3·3)	1·3 (0·7–2·3)	1·3 (0·6–2·3)	1·1 ↓ (0·6–2·0)
DRhigh+CD45RA- Tregs	0·3 (0·1–0·7)	0·2 (0·1–0·6)	0·2 (0·1–0·7)	0·2 (0·1–0·5)	0·2 ↓ (0–0·6)

↑↓Sgnificant differences were obtained compared to non-pregnant women.

The normal course of pregnancy is associated with changes in the composition of the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool with distinct T_reg cell subsets

In order to examine whether the reduced suppressive activity of the total CD4⁺CD127^low+/−CD25⁺Foxp3⁺ T_reg cell pool could be correlated with changes of its composition with distinct T_reg cell subsets, we monitored their percentages during the normal course of pregnancy and in the presence of characteristic gestation-associated diseases. To this end, PBMCs were additionally stained with anti-HLA-DR- and anti-CD45RA-specific antibodies and analysed by six-colour flow cytometric analysis. This resulted in the detection of four different T_reg subpopulations, as shown in Fig. 1d. The percentages of these T_reg cell subsets within the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool were estimated in the circulation of healthy pregnant women from first trimester on until term. Figure 3 and Table 1 show the changes of these four T_reg cell subsets during the normal course of pregnancy. The percentage of DR^low+CD45RA^- and DR^high+CD45RA^- T_reg cells (Fig. 3a,c) decreased significantly between the 10th and the 20th weeks of gestation and subsequently remained at the same level until term (Fig. 3b,d). Simultaneously, the percentage of naive DR^-CD45RA⁺ T_reg cells (Fig. 3e) increased significantly and also remained stable during the following course of pregnancy (Fig. 3f). The subset of DR^-CD45RA^- T_reg cells (Fig. 3g) did not show any changes during the normal course of pregnancy (Fig. 3h). Obviously, these changes in the composition of the CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool between the 10th and 20th weeks of gestation were not accompanied by changes in its suppressive activity (Fig. 2d–g). However, the suppressive activity of the total CD4⁺CD127^low+/−CD25⁺ T_reg cell pool decreased significantly near term, at a gestational age when no further changes in the composition of the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool could be detected. Such findings may indicate that, besides the composition of the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool with distinct T_reg cell subsets, further parameters may have an influence on its suppressive activity.

Fig. 3.

Detection of the percentages of the DR^low+CD45RA^-, DR^high+CD45RA^-, DR^-CD45RA^- and DR^-CD45RA⁺ T_reg subsets within the total CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3⁺) T_reg cell pool during the normal course of pregnancy. The figure shows the dot-blots of the gated DR^low+CD45RA^-, DR^high+CD45RA^-, DR^-CD45RA^- and DR^-CD45RA⁺ T_reg subsets and their quantitative changes during the normal course of pregnancy. The diagram presents the individual and median data obtained for non-pregnant (NP) and healthy pregnant women. The percentage of the DR^low+CD45RA^- T_reg subset (a,b) and of the DR^high+CD45RA^- T_reg subset (c,d) decreased significantly during the 10th and 20th weeks of gestation. In contrast, the percentage of the DR^-CD45RA⁺ T_reg subset increased during the same time (e,f). The percentage of the DR^-CD45RA^- T_reg subset did not show any significant changes during the whole course of pregnancy (g,h).

Pre-eclampsia is associated with an increased percentage of DR⁺CD45RA^- T_reg cells, while PL is associated with an increased percentage of DR^-CD45RA^- T_reg cells within both the total T_reg cell pool and the total CD4⁺ T cell pool

In order to examine whether the discrepancies concerning the suppressive potency of the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool between healthy and affected pregnancies could be correlated with its composition with the above-described distinct T_reg cell subsets (Fig. 1d), we assessed their percentages in pregnancies affected by pre-eclampsia (group 5), HELLP syndrome (group 6) CI (group 7) and PL (group 8), in comparison to healthy pregnancies in the third trimester (group 3 and group 4). Figure 4a–h show the quantitative alterations of these particular T_reg subsets in the presence of gestation-associated hypertensive diseases (pre-eclampsia and HELLP syndrome; Fig. 4a–d) and in the presence of preterm intrauterine activation (CI and PL; Fig. 4e–h). Figure 4i–l and Table 2 summarize the data obtained for all patient groups and present significant differences between healthy and affected pregnancies.

Fig. 4.

Detection of the percentages of the DR^low+CD45RA^-, DR^high+CD45RA^-, DR^-CD45RA^- and DR^-CD45RA⁺ T_reg subsets within the total CD4⁺CD127^low+/−CD25⁺forkhead box protein 3 (FoxP3⁺) T_reg cell pool in healthy and complicated pregnancies. The figure presents the individual data (a–h) and median values (i–l) obtained for pregnancies affected by pre-eclampsia () or HELLP syndrome () (a–d), and for pregnancies affected by CI () or PL () (e–h), in comparison to healthy pregnancies (HP) (♦). Pregnancies affected by pre-eclampsia revealed significantly increased percentages of the DR^low+CD45RA^- and DR^high+CD45RA^- T_reg subsets (a,b,i,j), while in pregnancies affected by HELLP syndrome only the DR^high+CD45RA^- T_reg subset was increased significantly (b,j). In contrast, the DR^-CD45RA^- and DR^low+CD45RA^- T_reg subsets were increased significantly in pregnancies affected by PL (e,g,i,k). The percentage of the naive DR^-CD45RA⁺ T_reg subset was decreased significantly in pregnancies affected by both pre-eclampsia and PL (d,h,l). CI: cervical insufficiency; PL: preterm labour necessitating preterm delivery; HELLP syndrome: haemolysis-elevated liver enzyme levels-low platelet count syndrome; MFI: mean fluorescence intensity.

Compared to healthy pregnancies, we found a significantly increased percentage of DR^low+CD45RA^- and DR^high+CD45RA^- T_regs in the T_reg pool of pregnancies affected by pre-eclampsia. However, pregnancies affected by HELLP syndrome did not show an increased percentage of DR^low+CD45RA^- T_reg cells, but had a significantly increased percentage of DR^high+CD45RA^- T_regs in their T_reg pool (Fig. 4a–b,i–j). In the presence of PL leading to preterm delivery, the percentages of DR^-CD45RA^- T_regs and of DR^low+CD45RA^- T_regs were increased significantly (Fig. 4e,i,g,k), while the occurrence of CI was not associated with changes in the percentages of these T_reg cell subsets (Fig. 4e–h,i–l). By contrast, we found that the presence of pre-eclampsia and PL, which were both associated with an increased percentage of DR^low+CD45RA^- T_regs, were accompanied with a significantly decreased percentage of naive DR^-CD45RA⁺ T_reg cells within their T_reg cell pool (Fig. 4d,h,l). As both patient collectives show a significantly reduced suppressive activity of their total CD4⁺CD127^low+/−CD25⁺ T_reg cell pool (Fig. 2d–g), it could be assumed that such changes could have an influence on its functional activity.

In order to examine whether the percentages of the different subsets within the total T_reg cell pool correspond to their absolute numbers within the total CD4⁺ T cell pool, their percentages were additionally calculated relative to the total CD4⁺ T cell pool. Figure 5 depicts the results obtained for these measurements. Overall, there was a significant decrease of the DR^low+CD45RA^- and DR^high+CD45RA^- T_reg subsets and a significant increase of the naive DR^-CD45RA⁺ T_regs subset during the normal course of pregnancy (Fig. 5). Compared to Fig. 4, exactly the same significant changes in the percentages of the different T_reg subsets between healthy and affected pregnancies were documented both for the total T_reg cell pool (Fig. 4) and the total CD4⁺ T cell pool (Fig. 5). Apparently, in the presence of complications such as pre-eclampsia and PL, DR^low+CD45RA^-, DR^high+CD45RA^- T_regs and DR^-CD45RA^- T_reg cells accumulated, while naive DR^-CD45RA⁺ T_reg cells declined within the CD4⁺ T cell pool. Such findings may propose that in the presence of pre-eclampsia or PL, the primarily existing naive DR^-CD45RA⁺ T_regs cell compartment shrinks because of an increased conversion into either DR^-CD45RA^- or DR⁺CD45RA^- T_reg cells. As even the total CD4⁺ T cell pool may vary during the normal course of pregnancy and between the different patient groups, we estimated its percentage within PBMCs isolated after Ficoll-Hypaque gradient centrifugation. We found a significantly decreased percentage of CD4⁺ T cells between non-pregnant and pregnant women, irrespective of the gestational age (Table 1). Compared to healthy pregnancies, the percentage of CD4⁺ T cells was reduced significantly (P < 0·01) in patients affected by PL, but was not different in patients with cervical insufficiency, pre-eclampsia or HELLP syndrome (Table 2).

Fig. 5.

Detection of the percentages of the DR^low+CD45RA^-, DR^high+CD45RA^-, DR^-CD45RA^- and DR^-CD45RA⁺ T_reg subsets within the total CD4⁺ T cell pool in healthy and complicated pregnancies. Concerning the changes in the percentages of the different T_reg subsets in healthy and complicated pregnancies, similar results were obtained for the percentages of the different T_reg subsets within both the total T_reg cell pool and the CD4⁺ T cell pool (see Fig. 4).

The DR^high+CD45RA^- T_reg cell subset is more suppressive than the pool of the residual T_reg cell subsets

Our data demonstrate that the suppressive activity of the total T_reg cell pool obtained from pregnancies affected by pre-eclampsia or PL is reduced significantly. Its composition in these patients is changed in the way that it contains a higher percentage of DR^low+CD45RA^- and DR^high+CD45RA^- T_regs in the case of pre-eclampsia and a higher percentage of DR^-CD45RA^- and DR^low+CD45RA^- T_regs in the case of PL. In contrast, its percentage of naive DR^-CD45RA⁺ T_regs is diminished strongly in both patient collectives (Table 2). It is known that DR expression on T_reg cells is associated with highly suppressive mature T_reg cells [17]. Therefore, it could be assumed that the suppressive activity of the T_reg pool depends mainly on the proportion of fully developed DR^high+CD45RA^- T_regs among the DR⁺CD45RA^- T_reg subset. Therefore, we examined whether these cells could have a potential effect of the suppressive activity of the total T_reg cell pool. For that, magnetically isolated T_reg cells obtained from three healthy, non-pregnant volunteers were stained with anti-CD4-, anti-CD25- and anti-HLA-DR-specific antibodies and sorted into a population of DR^high+CD45RA^- T_regs (Fig. 6a, R1) and into a population consisting of the remaining DR^low+CD45RA^-, DR^-CD45RA^- and naive DR^-CD45RA⁺ T_regs (Fig. 6a, R2). Subsequently, both T_reg populations were analysed concerning their suppressive activity (Fig. 6b). Figure 6b,c demonstrate clearly that both the maximum suppressive activity and the titre (T_reg/T_resp), with which a minimum suppressive activity of 15% was achieved, is increased strongly for the DR^high+CD45RA^- T_regs (R1) T_regs compared to the remaining T_reg population consisting of DR^low+CD45RA^-, DR^-CD45RA^- and naive DR^-CD45RA⁺ T_regs (R2).

Fig. 6.

Positive selection and functional testing of the DR^high+CD45RA^- T_reg subset. Magnetically isolated CD4⁺CD127^low+/−CD25⁺ T_reg were stained with anti-CD4, anti-CD25 and anti-human leucocyte antigen D-related (HLA-DR)-specific antibodies and sorted into a population of DR^high+CD45RA^- T_regs (a, R1) and into a population consisting of DR^low+CD45RA^-, DR^-CD45RA^- and naive DR^-CD45RA⁺ T_regs (a, R2). Subsequently, both T_reg populations obtained from three different subjects were analysed concerning their suppressive activity (b). The maximum suppressive activity (T_reg/T_resp = 1/1) and the minimum ratio of T_reg/T_resp (titre) with at least 15% suppression were increased strongly for the DR^high+CD45RA^- T_regs compared to the remaining T_regs (R2) (c).

Discussion

Currently, great efforts are being made to investigate the role of the immunosuppressive CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell compartment for both the normal and the abnormal course of pregnancy. In this study, we thoroughly assessed T_reg frequencies, their functional activity and their composition with distinct T_reg cell subsets (DR^low+CD45RA^-, DR^high+CD45RA^-, DR^-CD45RA^- and naive DR^-CD45RA⁺ T_regs) in the circulation of healthy and complicated pregnancies. Similar to our previous studies, when T_reg cells were simply detected on the basis of CD4, CD25 and FoxP3 [16] or on the basis of CD4, CD127 and CD25 [13], we observed that the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_regs among the total CD4⁺ T cell pool was decreasing during the normal course of pregnancy. Currently, such findings have been confirmed by other groups, although different gating strategies for the characterization of T_reg cells were used. Mjösberg et al. detected CD4^dim+CD25^high+ T_reg cells instead of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells, but also found decreased percentages in second-trimester women compared to non-pregnant women [11]. Tilburgs et al. detected CD4⁺CD25^high+ T_reg cells and found that their percentage of FoxP3⁺ and HLA-DR⁺ cells was decreased significantly in second- and third-trimester women compared to non-pregnant women [10]. An even increased percentage of such cells was found in the decidua basalis and in the decidua parietalis, indicating that a selective T_reg recruitment from the circulating pool into the placenta may occur, where these cells could act to prevent the acute allogeneic response towards the fetus [10].

With regard to functional studies of the circulating T_reg pool, we did not find significant differences between non-pregnant and healthy pregnant women. Its suppressive activity was preserved approximately until the 30th week of gestation. Changes in the composition of the total T_reg cell pool with the above-described T_reg cell subsets were observed between the 10th and 20th weeks of gestation. However, these changes were not associated with alterations of its suppressive activity. A significant decline of the suppressive activity could be detected near term. However, that decline was not associated with characteristic changes of its composition with different T_reg subsets. Therefore, further mechanisms may influence the functional activity of the T_reg compartment. Recently, Mjösberg et al. demonstrated that FoxP3 expression of T_reg cells could be influenced by the hormonal milieu and that it was already reduced in second-trimester women compared to non-pregnant women [11]. In particular, they were able to demonstrate that progesterone and 17β-oestradiol, which indeed reach maximum serum levels at the end of pregnancy, had the capacity to reduce FoxP3 expression of T_reg cells in vitro. However, other in-vitro examinations demonstrated recently that oestradiol rather potentiates the suppressive activity of CD4⁺CD25⁺ T_regs[21]. In addition, in-vivo assays, monitoring CD4⁺CD25⁺FoxP3⁺ T_reg cell numbers during the menstrual cycle, revealed tightly correlated increasing frequencies with increasing serum levels of oestradiol [22]. Moreover, most of these studies, supporting potential effects of hormones on the development, expansion and function of T_reg cells, were performed on mice [23]. Currently, it still remains unclear whether such findings can be transferred to the human system.

Table 2 summarize our data concerning the frequency, the functional activity and the composition of the total CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cell pool with distinct T_reg cell subsets in the presence of characteristic gestation-associated diseases compared to healthy pregnancies. We were not able to detect a decreased percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_reg cells within the total CD4⁺ T cell pool in the presence of pre-eclampsia, HELLP syndrome, CI or PL. In contrast, most recent articles documented decreased T_reg cell counts in patients affected by pre-eclampsia [14,16,24,25]. Such discrepancies may arise because human FoxP3 can be expressed transiently by in-vitro-activated T cells lacking regulatory function [26]. Recently, an inverse correlation between the expression of the IL-7 receptor α chain (CD127) and their suppressive function was shown for CD4⁺CD25⁺FoxP3⁺ T_reg cells [27,28]. Therefore, simultaneous assessment of CD127, CD25 and FoxP3 expression by CD4⁺ T cells may help to exclude non-T_reg cells and lead to accurate numbers of T_regs. Moreover, quantitative determinations of CD4⁺CD25^high+ T_reg cells in the absence of the FoxP3 marker confirm our results obtained for pregnancies affected by pre-eclampsia [29].

With regard to the functional activity of T_reg cells from affected pregnancies, we observed a significantly reduced suppressive activity for patients with pre-eclampsia and for patients with PL, while the suppressive potency of patients with HELLP syndrome or CI was in the normal range. Astonishingly, patients with pre-eclampsia and PL had a significantly reduced percentage of naive DR^-CD45RA⁺ T_regs within their total T_reg cell compartment. Simultaneously, an increased percentage of DR^high+CD45RA^- T_regs and DR^low+CD45RA^- T_regs was detected in patients with pre-eclampsia, while an increased percentage of DR^-CD45RA^- and DR^low+CD45RA^- T_regs was found in patients with PL. Identical results were obtained concerning the percentages of these T_reg cell subsets within the total CD4⁺ T cell pool, in fact, during the course of normal pregnancy and in the presence of pre-eclampsia or PL. Such findings suggest that naive DR^-CD45RA⁺ T_reg cells may be increasingly released by the thymus during the normal course of pregnancy, and that these cells may be converted into DR^-CD45RA^- DR^low+CD45RA^- and DR^high+CD45RA^- T_regs in the presence of these diseases. Currently, we do not know whether the decrease of the naive DR^-CD45RA⁺ T_reg population contributes to the impaired suppressive activity of the total CD4⁺CD127⁺CD25⁺ T_reg cell pool. So far, it is merely documented that naive RA⁺ T_reg cells are more prevalent in early life [30]. Highest percentages were detected in the cord blood, while in adults the total T_reg pool contained mainly RO⁺ memory T_regs[30]. Differences of the suppressive activity between naive RA⁺ T_regs and memory RO⁺ T_regs were not ascertained [31]. However, it was shown recently that the extent of the suppressive activity of RA⁺ T_regs depends decisively on its proportion of cells co-expressing CD31, a distinct T_reg subset that enters the circulation as recent thymic emigrants [32]. Therefore, it may be possible that the suppressive capacity of naive DR^-CD45RA⁺ T_regs may be different in pregnant and non-pregnant women.

Recently, we demonstrated that particularly the subset of DR⁺ T_regs contributes essentially to the suppressive activity of the total T_reg cell pool [13]. Thereby, we showed that the reduced suppressive activity of T_reg cells observed in patients with PL was not associated with a decreased percentage of DR⁺ T_reg cells, but that the level of HLA-DR expression of their DR⁺ T_reg cells (HLA-DR MFI) was strongly reduced [13]. Our current data clearly demonstrate that the percentage of DR^low+CD45RA^- T_regs is increased significantly in these patients. As the percentage of DR^-CD45RA^- T_regs was also increased, one could assume that these patients could have deficiencies in the maturation of DR^-CD45RA^- T_regs into highly suppressive DR^high+CD45RA^- T_regs.

Astonishingly, we found that pre-eclamptic women, whose T_reg pool and CD4⁺ T cell pool contained an increased percentage of DR^high+CD45RA^- T_regs and DR^low+CD45RA^- T_regs concurrently exhibited a reduced suppressive activity. In contrast, the suppressive activity of the T_reg pool in patients with HELLP syndrome was in the normal range, but contained an increased percentage of the DR^high+CD45RA^- T_regs. Obviously, the pre-eclamptic patients have deficiencies in the functional activity of their T_reg pool, although it contains a high proportion of HLA-DR⁺ T_regs. Probably, in patients with HELLP syndrome the high proportion of fully developed, highly suppressive DR^high+CD45RA^- T_regs compensates this loss of suppressive activity observed in pre-eclamptic patients. It could also be possible that the responder T cells in pre-eclamptic patients could be resistant to T_reg suppression. In either case, it seems that the DR^high+CD45RA^- T_reg cells contribute potentially to the suppressive activity of the total T_reg cell pool. Therefore, we examined the role of this T_reg subset for the suppressive activity of the total T_reg cell pool. Positive selection and analysis of its suppressive activity in comparison to the remaining T_reg cells revealed that DR^high+CD45RA^- T_reg cells are more suppressive than the pool of the residual T_reg cells. Further studies may be necessary to evaluate the contribution of each of these T_reg subsets to the suppressive activity of the total T_reg cell pool.

Disclosure

None of the authors has any conflict of interest related to this manuscript.