Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (µMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

Maternal B cell signaling orchestrates fetal development in mice.

B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4 mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.

A minigene DNA vaccine encoding peptide epitopes derived from Galectin-1 has protective antitumoral effects in a model of neuroblastoma.

We recently identified Galectin-1 (Gal-1), a ß-galactoside-binding lectin, as a novel immune regulator in neuroblastoma (NB). Here, we characterized the tolerogenic function of Gal-1 within the CD8+ T cell compartment and further evaluated its relevance as an antigen for effective DNA vaccination against NB in a mouse model. NB cells with Gal-1 knockdown (NXS-2L) exhibited significantly reduced tumor growth compared to NXS-2 NB cells. Administration of anti-CD8 antibodies prevented this antitumor effect, with primary tumor growth comparable to that from Gal-1 (G1)-sufficient NB cells. Peptide epitope screening with online databases and in silico docking experiments predicted the sequences "FDQADLTI" (#1), "GDFKIKCV" (#2), and "AHGDANTI" (#3) to have superior H2-KK binding affinities and "KFPNRLNM" (#4), "DGDFKIKCV" (#5), and "LGKDSNNL" (#6) to have superior H2-DD binding affinities. Minigenes encoding G1-KK (#1-#2-#3), G1-DD (#4-#5-#6) and the triplet with the highest affinity, G1-H (#1-#2-#4), were generated and cloned into a ubiquitin-containing plasmid (pU). Mice receiving pU-G1-KK or pU-G-1H presented a reduction in the s.c. tumor volume and weight of up to 80% compared to control mice; this reduction was associated with increased cytotoxicity of isolated splenocytes from vaccinated animals. Vaccination with pUG1-DD showed a lower capability to suppress primary tumor progression. In conclusion, Gal-1 expression by NB negatively regulates CD8+ T cells. Vaccination with DNA plasmids encoding Gal-1 epitopes overcomes immune escape, enhances CD8+ T cell-dependent immunity and displays effective antitumor activity against NB.

Using ultrasound to define the time point of intrauterine growth retardation in a mouse model of heme oxygenase-1 deficiency†.

The enzyme heme oxygenase-1 (HO-1), encoded by the HMOX1 gene, mediates heme catabolism by cleaving free heme. We have previously revealed the importance of HO-1 in pregnancy. Here, we determined the impact of maternal or paternal HO-1 deficiency on fetal growth and placental parameters throughout gestation. We mated Hmox1-sufficient (WT), partial (HET)-, or total (KO)-deficient BALB/c female mice with Hmox1-WT or -KO BALB/c males and performed ultrasound analysis to monitor placental and fetal growth. Doppler measurements were used to determine maternal blood flow parameters. Offspring weights and feto-placental indices (FPI) were also determined. We found a significantly increased number of underdeveloped fetuses at gd10 in HET females that were mated with WT males compared with WT × WT pairings. At the same gestational age, underdeveloped placentas could be detected in HET females mated with KO males. Many fetuses from the KO × KO combination died in utero between gd12 and gd14. At gd14, abnormal placental parameters were found in surviving fetuses, which had significant reduced weights. Moreover, only 3.11% female and 5.33% male KO pups resulted from 10 HET × HET breeding pairs over 1 year. Our results show that HO-1 from both maternal and paternal origins is important for proper placental and fetal growth. Placental growth restriction and occurrence of abortions in mice that were partially or totally deficient in HO-1 were recorded in vivo from gd10 onwards. Future studies will focus on elucidating the cellular and molecular mechanisms behind these observations.

Human Miscarriage Is Associated With Dysregulations in Peripheral Blood-Derived Myeloid Dendritic Cell Subsets.

Dendritic cells (DC) are critically involved in decisions related to the acceptance or rejection of the foreign fetal antigens by the maternal immune system. However, particularly for human peripheral blood DCs (PBDC), available literature is rather inconsistent and the factors regulating these cells are ill-defined. Here, we investigated the phenotype and functionality of different human PBDC subsets during normal and pathologic pregnancies and studied an involvement of human chorionic gonadotropin (hCG) in PBDC regulation. Peripheral blood samples were obtained from normal pregnant women in all three trimesters, from first trimester miscarriage patients and from healthy non-pregnant women. Samples were analyzed for plasma hCG levels, for regulatory T (Treg) cell numbers, for frequencies of total and mature plasmacytoid (PDC) and myeloid (MDC1 and MDC2) PBDC subsets and for their cytokine secretion. In vitro assays, culturing PDC, MDC1 or MDC2 in the presence of two trophoblast cell lines, placenta explant supernatants or two hCG preparations were performed. The Treg-inducing capability of hCG- or non-hCG-treated stimulated MDC1 was assessed. Total and mature MDC1 and MDC2 frequencies increased during the first and second trimester of normal pregnancy, respectively. Miscarriage was associated with a reduced MDC1 and an increased MDC2 activation profile. PDC were not altered neither during normal pregnancy progression nor during miscarriage. In vitro, the culture of isolated PBDC subsets in the presence of placenta-derived factors impaired the maturation of MDC1 and differentially affected PDC maturation. An inhibitory effect on MDC1 and PDC maturation was also proven for the urine-derived hCG preparation. Finally, we observed a Treg cell elevation during early normal pregnancy that was not present in miscarriages. Stimulated MDC1 induced Treg cells in vitro, however, hCG was not involved in this process. Our findings suggest that during normal pregnancy PBDC subsets are differentially regulated dependent on gestational age. Miscarriage seems to be associated with dysregulations in the myeloid PBDC subsets and with disturbances in Treg cell frequencies. Moreover, our results propose an interdependency between MDC1 and Treg cells during early pregnancy. hCG, although shown to impair MDC1 maturation, does not seem to be a key regulator of PBDC alterations during pregnancy.

IL-10 producing B cells rescue mouse fetuses from inflammation-driven fetal death and are able to modulate T cell immune responses.

Understanding the mechanisms leading to fetal death following maternal subclinical infections is crucial to develop new therapeutic strategies. Here we addressed the relevance of IL-10 secreting B cells (B10) in the maintenance of the immune balance during gestation. µMT females lacking mature B cells presented normal pregnancies, although their fetuses were smaller and their Treg pool did not expand as in B cell sufficient controls. Pregnant µMT females were more susceptible to LPS despite having less Treg; their fetuses died at doses compatible with pregnancy in WT animals. Adoptive transfer of IL-10 negative B effector cells or B cells from IL-10 deficient mice did not modify this outcome. The transfer of B10 cells or application of recombinant murine IL-10 reduced the fetal loss, associated with a normalization of Treg numbers and cytokine modulation at the feto-maternal interface. B cell-derived IL-10 suppressed the production of IL-17A and IL-6 by T cells and promoted the conversion of naïve cells into Treg. B10 cells are required to restore the immune balance at the feto-maternal interface when perturbed by inflammatory signals. Our data position B cells in a central role in the maintenance of the balance between immunity and tolerance during pregnancy.

Immune Modulatory Effects of Human Chorionic Gonadotropin on Dendritic Cells Supporting Fetal Survival in Murine Pregnancy.

Dendritic cells (DCs) are critically involved in the determination of immunity vs. tolerance. Hence, DCs are key regulators of immune responses either favoring or disfavoring fetal survival. Several factors were proposed to modulate DC phenotype and function during pregnancy. Here, we studied whether the pregnancy hormone human chorionic gonadotropin (hCG) is involved in DC regulation. In vitro, bone marrow-derived DCs (BMDCs) were stimulated in the presence or absence of urine-purified or recombinant hCG (rhCG) preparations. Subsequently, BMDC maturation was assessed. Cytokine secretion of activated BMDCs and their capability to enforce TH1, TH2, TH17, or Treg cell differentiation was determined after rhCG treatment. Moreover, the in vivo potential of hCG-modulated BMDCs to influence pregnancy outcome, Treg cell number, and local cytokine expression was evaluated after adoptive transfer in a murine abortion-prone model before and after conception. Both hCG preparations impaired the maturation process of BMDCs. rhCG treatment did neither alter cytokine secretion by BMDCs nor their ability to drive TH1, TH2, or TH17 differentiation. rhCG-treated BMDCs augmented the number of Treg cells within the T cell population. Adoptive transfer of rhCG-treated BMDCs after conception did not influence pregnancy outcome. However, transfer of hCG-treated BMDCs prior to mating had a protective effect on pregnancy. This positive effect was accompanied by increased Treg cell numbers and decidual IL-10 and TGF-ß expression. Our results unveil the importance of hCG in retaining DCs in a tolerogenic state, thereby promoting Treg cell increment and supporting fetal survival.

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.

Exploring the potential of low doses carbon monoxide as therapy in pregnancy complications.

Heme Oxygenase-1 (HO-1) has been shown to play a pivotal role in pregnancy outcome and its ablation leads to abnormal placentation, intrauterine fetal growth restriction (IUGR) and subsequent intrauterine fetal death. Carbon monoxide (CO) has been found to mimic the protective effects of HO-1 activity, rescuing HO-1-deficient fetuses. This gasotransmitter arises in biological systems during the oxidative catabolism of heme by HO. Here, we explored the potential of CO in preventing IUGR and established the optimal doses and therapeutic time window in a clinically relevant mouse model. We additionally investigated the pathways activated upon CO application in vivo. We established 50 ppm as the best lowest dose of CO necessary to prevent growth restriction being the optimal time frame during days 3 to 8 of mouse pregnancy. CO lead to higher fetal and placental weights and avoided fetal death without showing any pathologic effects. CO breathing further suppressed inflammatory responses, diminished placenta apoptosis and complement deposition and regulated placental angiogenesis. Our results confirm the protective role of the HO-1/CO axis and point this gas as an emerging therapeutic possibility which is worth to further explore.

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.

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-beta 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.

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. METHOD OF STUDY: We included in our studies the well-known CBA/JxDBA/2J abortion-prone combination using CBA/JxBALB/c as controls. CBA/JxC57/BL6 and BALB/cxC57/BL6 were included as further controls. Animals were killed on days 0, 2, 5, 8, 10, and 12 of pregnancy to measure the levels of Treg, pregnancy-related hormones and IDO expression. RESULTS: A Treg augmentation in normal pregnancy combinations could be observed on day 2 in several organs contrary to the observations made in abortion-prone mice. No differences in hormonal levels could be seen among all groups. IDO was expressed exclusively in placenta starting from day eight, showing no variations among the groups. CONCLUSION: Differences in Treg levels and pregnancy outcome do not correlate with changes in hormonal levels. In addition, as Treg augmentation takes place early and it is observed mainly in the decidual component of the fetal-maternal interface, IDO does not seem to be the pathway underlying Treg protective activity as proposed for humans.