Lipid phosphatases identified by screening a mouse phosphatase shRNA library regulate T-cell differentiation and protein kinase B AKT signaling.

Screening a complete mouse phosphatase lentiviral shRNA library using high-throughput sequencing revealed several phosphatases that regulate CD4 T-cell differentiation. We concentrated on two lipid phosphatases, the myotubularin-related protein (MTMR)9 and -7. Silencing MTMR9 by shRNA or siRNA resulted in enhanced T-helper (Th)1 differentiation and increased Th1 protein kinase B (PKB)/AKT phosphorylation while silencing MTMR7 caused increased Th2 and Th17 differentiation and increased AKT phosphorylation in these cells. Irradiated mice reconstituted with MTMR9 shRNA-transduced bone marrow cells had an elevated proportion of T-box transcription factor T-bet expressors among their CD4 T cells. After adoptive transfer of naïve cells from such reconstituted mice, immunization resulted in a greater proportion of T-box transcription factor T-bet-expressing cells. Thus, myotubularin-related proteins have a role in controlling in vitro and in vivo Th-cell differentiation, possibly through regulation of phosphatidylinositol [3,4,5]-trisphosphate activity.

Analysis of naïve lung CD4 T cells provides evidence of functional lung to lymph node migration.

The proportion of CD4 T cells with phenotypic and functional properties of naïve cells out of total CD4 T cells is similar in the lung parenchyma and lymph nodes. On treatment with a sphingosine-1-phosphate agonist, the frequency of these cells falls precipitously, but with a delay of ∼14 h compared with blood CD4 T cells; neither anti-CD62L nor pertussis toxin prevents entry of naïve CD4 T cells into the lung. Based on treatment with anti-CD62L and the use of CCR7(-/-) cells, lung naïve CD4 T cells appear to migrate to the mediastinal lymph nodes along a CD62L-independent, CCR7-dependent pathway. Cells that have entered the node in this manner are competent to respond to antigen. Thus, a portion (approximately one-half) of naïve CD4 T cells appears to enter the mediastinal lymph nodes through a blood-to-lung-to-lymph node route.

Transplantation tolerance to a single noninherited MHC class I maternal alloantigen studied in a TCR-transgenic mouse model.

The mechanisms underlying tolerance to noninherited maternal Ags (NIMA) are not fully understood. In this study, we designed a double-transgenic model in which all the offspring's CD8(+) T cells corresponded to a single clone recognizing the K(b) MHC class I protein. In contrast, the mother and the father of the offspring differed by the expression of a single Ag, K(b), that served as NIMA. We investigated the influence of NIMA exposure on the offspring thymic T cell selection during ontogeny and on its peripheral T cell response during adulthood. We observed that anti-K(b) thymocytes were exposed to NIMA and became activated during fetal life but were not deleted. Strikingly, adult mice exposed to NIMA accepted permanently K(b+) heart allografts despite the presence of normal levels of anti-K(b) TCR transgenic T cells. Transplant tolerance was associated with a lack of a proinflammatory alloreactive T cell response and an activation/expansion of T cells producing IL-4 and IL-10. In addition, we observed that tolerance to NIMA K(b) was abrogated via depletion of CD4(+) but not CD8(+) T cells and could be transferred to naive nonexposed mice via adoptive transfer of CD4(+)CD25(high) T cell expressing Foxp3 isolated from NIMA mice.

Tolerance induction to self-MHC antigens in fetal and neonatal mouse B cells.

We have studied the mechanisms of tolerance induction to self-MHC antigens in mouse B cells during fetal development and the post-natal period. To monitor the fate of autoreactive B cell clones, we used the 3-83 micro delta B cell receptor (BCR)-transgenic (Tg) and -knock-in (KI) mouse models. These BCR-Tg and -KI B cells recognize the MHC class I molecules H-2K(k) and H-2K(b), with a high or moderate affinity, respectively. We compared the fate of BCR-Tg and -KI B cells in H-2K(b)-bearing animals and H-2K(b)-negative controls at various stages of their fetal development and post-natal life. Our data show that, in contrast to what occurs in adult B cells, anergy is the main component of tolerance induction in 3-83 micro delta BCR-Tg K(b+) autoreactive fetuses, while 3-83 BCR-KI fetuses primarily use receptor editing. Interestingly, autoreactive B cell deletion is absent or merely marginal before birth. Our results indicate that tolerance induction is effective as early as embryonic day 16.5 and that in the fetus and neonate, like in the adult, the main mechanism of B cell tolerance functioning in the 3-83 KI system is receptor editing. In contrast, in the 3-83 micro delta mice where receptor editing is hindered, adult and fetal B cells differ in their preferential use of mechanisms leading to self-tolerance (i.e. deletion versus anergy).

Affinity-dependent alterations of mouse B cell development by noninherited maternal antigen.

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 mu/delta transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2Kk and H-2Kb, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2Kk or H-2Kb maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2Kk-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2Kb-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.

Pregnancy-induced alterations of B cell maturation and survival are differentially affected by Fas and Bcl-2, independently of BcR expression.

In the present work, we have analyzed the roles of two molecules involved in the regulation of cell survival, Bcl2 and Fas, in the pregnancy-induced down-regulation of B lymphopoiesis in mice. Our results show that the overexpression of the anti-apoptotic molecule Bcl2 in Bcl2-transgenic (Tg) B cells is able to protect 'D' fraction pre-B cells from pregnancy-induced deletion. In contrast, in Fas(lpr/lpr) mice bearing a mutated cell death receptor Fas, such B cell targets are not protected. Moreover, bone marrow B cell sub-populations at both ends of the differentiation pathway, i.e. pre-pro 'A' and mature 'E-F' fraction B cells, which are not the major targets of the pregnancy-induced down-regulation, are doubled during pregnancy in Fas(lpr/lpr) mice only. Altogether, these data strongly suggest that B cell down-regulation during pregnancy is due to apoptotic events blocked by Bcl2, but does not depend on a functional Fas receptor. The expression of a transgenic BcR in the 3-83mudelta BcR-Tg mouse model yields similar observations, which indicates that early BcR expression does not alter bone marrow B cell fates during pregnancy.

Tolerance of the fetus by the maternal immune system: role of inflammatory mediators at the feto-maternal interface.

The adaptive immune system of placental mammals has evolved to tolerate the fetus. Rejection of the fetus by adaptive immune responses is therefore a rare event, with abortion being caused more frequently by inflammation in the placenta. This review will cover recent aspects of immune privilege and the innate immune system at the feto-maternal interface, citing examples of the role played by microbial infections in fetal demise.

Placental anomalies and fetal loss in mice, after administration of doxycycline in food for tet-system activation.

During the course of a study aiming to obtain a tetracycline (Tet)-inducible transgene expression restricted to the placenta, we have observed a toxicity of doxycycline (dox) given in the food at doses of 2.5-10 mg/g to pregnant mice from two different inbred strains. During the second half of gestation, dox-fed non-transgenic mice presented placental anomalies and impaired fetal development proportional to the dose of antibiotic. Thus, dox administered in commonly used food doses can have an adverse effect on pregnancy. These observations are important for studies of placental or fetal development using inducible gene promoters.

At the innate frontiers between mother and fetus: linking abortion with complement activation.

The intricate mechanisms regulating fetomaternal interactions are still largely uncharacterized. Recent papers have revealed a major role for the innate immune system during abortion. Different experimental conditions-deletion of a complement regulator, injection of anti-phospholipid antibodies into mothers, or allo-recognition of fetuses in the presence of an IDO inhibitor-all lead to complement activation, inflammation, and fetal loss. These observations also raise new questions on the relationship between the adaptive and innate systems during pregnancy.