Stefan Fest

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.

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.

Questioning the Th1/Th2 Paradigm in Reproduction: Peripheral Levels of IL-12 are Down-Regulated in Miscarriage Patients

Zenclussen AC, Fest S, Busse P, Joachim R, Klapp BF, Arck PC. Questioning the Th1/Th2 paradigm in reproduction: peripheral levels of IL‐12 are down‐regulated in miscarriage patients. AJRI 2002; 48:245–251

Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide

Pregnancy establishment implies the existence of a highly vascularized and transient organ, the placenta, which ensures oxygen supply to the fetus via haemoproteins. Haem metabolism, including its catabolism by haem oxygenase‐1 (HO‐1), should be of importance in maintaining the homeostasis of haemoproteins and controlling the deleterious effects associated with haem release from maternal or fetal haemoglobins, thus ensuring placental function and fetal development. We demonstrate that HO‐1 expression is essential to promote placental function and fetal development, thus determining the success of pregnancy. Hmox1 deletion in mice has pathological consequences for pregnancy, namely suboptimal placentation followed by intrauterine fetal growth restriction (IUGR) and fetal lethality. These pathological effects can be mimicked by administration of exogenous haem in wild‐type mice. Fetal and maternal HO‐1 is required to prevent post‐implantation fetal loss through a mechanism that acts independently of maternal adaptive immunity and hormones. The protective HO‐1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of haem catabolism by HO‐1 that restores placentation and fetal growth. In a clinical relevant model of IUGR, CO reduces the levels of free haem in circulation and prevents fetal death. We unravel a novel physiological role for HO‐1/CO in sustaining pregnancy which aids in understanding the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy pathologies. Copyright

Neuroblastoma triggers an immunoevasive program involving galectin-1-dependent modulation of T cell and dendritic cell compartments

The immunosuppressive strategies devised by neuroblastoma (NB), the most common solid extracranial childhood cancer, are poorly understood. Here, we identified an immunoevasive program triggered by NB through secretion of galectin‐1 (Gal‐1), a multifunctional glycan‐binding protein. Human and mouse NB cells express and secrete Gal‐1, which negatively regulates T cell and dendritic cell function. When injected subcutaneously in syngeneic A/J mice, knockdown transfectants expressing low amounts of Gal‐1 (NXS2/L) showed reduction of primary tumor growth by 83–90% and prevented spontaneous liver metastases in contrast to NXS2 cell variants (NXS2/H, NXS2 wildtype) expressing high amounts of Gal‐1. Splenocytes from mice receiving Gal‐1 knockdown NXS2/L cells secreted higher amounts of IFN‐γ and displayed enhanced cytotoxic T‐cell function compared to NXS2/H or NXS2 controls. Immunohistochemical analysis revealed a six‐ to tenfold increase in the frequency of CD4+ and CD8+ T cells infiltrating tumors from mice receiving knockdown transfectants. This effect was confirmed by in vitro migration assays. Finally, supernatants of NXS2/H or NXS2 cells suppressed dendritic cell (DC) maturation and induce T cell apoptosis, whereas these effects were only marginal on DCs and T cells exposed to supernatants from NXS2/L cells. These results demonstrate a novel immunoinhibitory role of the Gal‐1‐glycan axis in NB, highlighting an alternative target for novel immunotherapeutic modalities.

Survivin minigene DNA vaccination is effective against neuroblastoma.

The inhibitor of apoptosis protein survivin is highly expressed in neuroblastoma (NB) and survivin‐specific T cells were identified in Stage 4 patients. Therefore, we generated a novel survivin minigene DNA vaccine (pUS‐high) encoding exclusively for survivin‐derived peptides with superior MHC class I (H2‐Kk) binding affinities and tested its efficacy to suppress tumor growth and metastases in a syngeneic NB mouse model. Vaccination was performed by oral gavage of attenuated Salmonella typhimurium SL7207 carrying pUS‐high. Mice receiving the pUS‐high in the prophylactic setting presented a 48–52% reduction in s.c. tumor volume, weight and liver metastasis level in contrast to empty vector controls. This response was as effective as a survivin full‐length vaccine and was associated with an increased target cell lysis, increased presence of CD8+ T‐cells at the primary tumor site and enhanced production of proinflammatory cytokines by systemic CD8+ T cells. Furthermore, depletion of CD8+ but not CD4+ T‐cells completely abrogated the pUS‐high mediated primary tumor growth suppression, demonstrating a CD8+ T‐cell mediated effect. Therapeutic vaccination with pUS‐high led to complete NB eradication in over 50% of immunized mice and surviving mice showed an over 80% reduction in primary tumor growth upon rechallenge in contrast to controls. In summary, survivin‐based DNA vaccination is effective against NB and the rational minigene design provides a promising approach to circumvent potentially hazardous effects of using full length antiapoptotic genes as DNA vaccines.

Blockage of Heme Oxygenase-1 Abrogates the Protective Effect of Regulatory T Cells on Murine Pregnancy and Promotes the Maturation of Dendritic Cells

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

Mast cells rescue implantation defects caused by c-kit deficiency

Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. KitW-sh/W-sh mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-β and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.

The persistence of paternal antigens in the maternal body is involved in regulatory T-cell expansion and fetal-maternal tolerance in murine pregnancy.

Citation Zenclussen ML, Thuere C, Ahmad N, Wafula PO, Fest S, Teles A, Leber A, Casalis PA, Bechmann I, Priller J, Volk H‐D, Zenclussen AC. The persistence of paternal antigens in the maternal body is involved in regulatory T‐cell expansion and fetal‐maternal tolerance in murine pregnancy. Am J Reprod Immunol 2010; 63: 200–208