Ana Claudia Zenclussen

Abnormal T-cell reactivity against paternal antigens in spontaneous abortion: adoptive transfer of pregnancy-induced CD4+CD25+ T regulatory cells prevents fetal rejection in a murine abortion model.

Mammalian pregnancy is thought to be a state of immunological tolerance. The mechanisms underlying this phenomenon are still poorly understood. Here, we determined whether an inappropriate function of T regulatory (Treg) cells is involved in the pathogenesis of spontaneous abortion. We evaluated spleen and decidual lymphocytes from CBA/J mice undergoing immunological abortion (DBA/2J-mated) or having normal pregnancy (BALB/c-mated) on day 14 of gestation for ex vivo cytokine production after PMA or paternal antigen (alloantigen) stimulation. Treg activity was characterized by quantifying CD4(+)CD25(+) cells, foxp3 expression, and interleukin-10 secretion. Decidual lymphocytes from abortion CBA/J mice contained a significantly higher frequency of interferon-gamma-producing T cells specific for paternal antigens compared to those from normal pregnancy (7.8% versus 2.7%, P < 0.05). Compared to virgin CBA/J females, normal pregnant mice showed strongly elevated numbers of CD4(+)CD25(+) and interleukin-10(+) Treg cells in the thymus whereas significantly lower frequencies of Treg cells were observed in abortion mice. Very interestingly, CD4(+)CD25(+) Treg cells from normal pregnant and nonpregnant CBA/J mice could inhibit both proliferation and interferon-gamma secretion of lymphocytes from abortion mice in vitro whereas in vivo prevention of fetal rejection could only be achieved after adoptive transfer of Treg cells from normal pregnant mice. Our data suggest that pregnancy-induced Treg cells play a vital role in maternal tolerance to the allogeneic fetus.

Human Chorionic Gonadotropin Attracts Regulatory T Cells into the Fetal-Maternal Interface during Early Human Pregnancy

Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-β mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.

Regulatory T cells induce a privileged tolerant microenvironment at the fetal-maternal interface.

The mechanisms underlying immune tolerance during pregnancy are poorly understood. In this regard, Treg seem to play an important role in mediating maternal tolerance to the fetus. We proposed a crucial role of T regulatory cells (Treg) in avoiding immunological rejection of the fetus after observing diminished number and function of Treg in abortion‐prone mice. We further confirmed the protective role of Treg during pregnancy by transferring pregnancy‐induced Treg into abortion‐prone mice, which prevented rejection. Here, we analyzed the mechanisms involved in Treg‐mediated protection. As expected, Treg therapy prevented abortion, while expanding the peripheral and thymic Treg population. Surprisingly, the decidual levels of the Th1 cytokines IFN‐γ and TNF‐α were not diminished after therapy. Interestingly, the mRNA levels of leukemia inhibitory factor, TGF‐β and heme oxygenase‐1 at the fetal‐maternal interface were dramatically up‐regulated after Treg transfer, while the levels of indolamine 2,3‐dioxygenase remained unchanged. Our data suggest that Treg treatment can not prevent T cell infiltration or high Th1 levels but is able to create a privileged tolerant microenvironment at the fetal‐maternal interface, further shedding light onto the molecular mechanisms involved in pregnancy tolerance.

The progesterone derivative dydrogesterone abrogates murine stress-triggered abortion by inducing a Th2 biased local immune response

Stress is known to induce abortions in mice and humans, putatively via increased levels of abortogenic Th1 cytokines and a decrease of progesterone. Adequate levels of progesterone exert an antiabortive response through binding to the progesterone-receptor, which induces the release of progesterone-induced blocking factor (PIBF) from lymphocytes. PIBF is highly pregnancy-protective by induction of a Th2 biased immune activity. The aim of this study was to investigate the effect of the progesterone derivative dydrogesterone (6-dehydro-retroprogesterone) in stress-triggered murine abortion. DBA/2J-mated CBA/J female mice were randomized in different groups: two groups were treated with different dydrogesterone dosages in a single injection before exposure to sound stress on Day 5 of pregnancy, one group was exposed to stress without dydrogesterone treatment, the fourth group received no stress and no dydrogesterone. On gestation Day 13, a highly elevated abortion rate was detected in stressed mice compared to control mice. Stressed animals presented lower levels of progesterone and PIBF in plasma and a reduced staining intensity of progesterone receptor at the feto-maternal interface. Injection of dydrogesterone abrogated the effect of stress on the abortion rate. Further, dydrogesterone increased levels of plasma PIBF in stressed mice, but did not affect progesterone levels. Interestingly, dydrogesterone dramatically increased the percentage of IL-4 positive decidual immune cells in stressed mice. Our data suggest that dydrogesterone abrogates stress-triggered abortion by inducing a Th2 biased local immune response.

Regulatory T cells and their role in pregnancy.

Citation Leber A, Teles A, Zenclussen AC. Regulatory T cells and their role in pregnancy. Am J Reprod Immunol 2010

REVIEW ARTICLE: Regulatory T Cells and Their Role in Pregnancy

Citation Leber A, Teles A, Zenclussen AC. Regulatory T cells and their role in pregnancy. Am J Reprod Immunol 2010

Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies.

Problem:  We previously reported a diminished expression of the heme‐degrading enzymes heme oxygenases (HO)‐1 and HO‐2 in decidua and placenta from mice undergoing Th1‐mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO‐1 and HO‐2 is also reduced at the feto‐maternal interface of pathologic human pregnancies.

Kinetics of regulatory T cells during murine pregnancy.

Problem  The semi‐allogeneic fetus is usually tolerated by the maternal immune system. This was proposed to be modulated by CD4+CD25+foxp3+ regulatory T cells (Treg). We aimed to determine the kinetics of Treg during murine gestation and investigate whether changes in Treg levels respond to hormonal variations during pregnancy or generated changes in the local indolamine dioxygenase (IDO) expression.

Introducing a mouse model for pre-eclampsia: adoptive transfer of activated Th1 cells leads to pre-eclampsia-like symptoms exclusively in pregnant mice

Pre‐eclampsia (PE) is the most severe pregnancy‐related disease, leading to high maternal and fetal morbidity/mortality. Immunological imbalances associated with endothelial cell dysfunction have been hypothesized as a cause for the onset and perpetuation of PE. Valid and reliable animal models are urgently required to test this hypothesis and to better understand the mechanisms underlying PE. We developed a novel PE‐model by adoptively transferring activated BALB/c Th1‐like splenocytes into allogeneically pregnant BALB/c female mice during late gestation; the model mimicked the symptoms of PE, i.e. increased blood pressure and glomerulonephritis accompanied by proteinuria. Interestingly, these PE‐like symptoms were not detectable in non‐pregnant recipients of activated Th1‐like cells. Adoptive cell transfer adversely affected the outcome of pregnancy by increasing fetal rejection, with uterine immune cells showing an inflammatory profile. In conclusion, we have established a valid and reliable PE mouse model, which opens vast opportunities for therapeutic interventions.

Pre‐eclampsia is not Associated with Changes in the Levels of Regulatory T Cells in Peripheral Blood

The acceptance of the semi‐allogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, regulatory T cells (Tregs) were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile, thus preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Treg. The major aim of the present work was to investigate if pre‐eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks.