Ana Cumano

A novel proteolytic cleavage involved in Notch signaling : the role of the disintegrin-metalloprotease TACE

The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway.

Jak2 Deficiency Defines an EssentialDevelopmental Checkpoint in DefinitiveHematopoiesis

Janus kinases (Jaks) play an important role in signal transduction via cytokine and growth factor receptors. A targeted inactivation of Jak2 was performed. Jak2-/- embryos are anemic and die around day 12.5 postcoitum. Primitive erythrocytes are found, but definitive erythropoiesis is absent. Compared to erythropoietin receptor-deficient mice, the phenotype of Jak2 deficiency is more severe. Fetal liver BFU-E and CFU-E colonies are completely absent. However, multilineage hematopoietic stem cells (CD34low, c-kit(pos)) can be found, and B lymphopoiesis appears intact. In contrast to IFNalpha stimulation, Jak2-/- cells do not respond to IFNgamma. Jak2-/- embryonic stem cells are competent for LIF signaling. The data provided demonstrate that Jak2 has pivotal functions for signal transduction of a set of cytokine receptors required in definitive erythropoiesis.

CD25+ CD4+ T Cells Regulate the Expansion of Peripheral CD4 T Cells Through the Production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25+ subpopulation of naturally activated (CD45RBlow) CD4 T cells, but not CD25− CD45RBlow CD4 T cells, inhibits the accumulation of cotransferred CD45RBhigh CD4 T cells in lymphocyte-deficient mice. However, both CD25+ and CD25− CD45RBlow CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4+ cells, we conclude that expansion control and disease prevention are largely independent processes. CD25+ CD45RBlow CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RBhigh CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25+ CD45RBlow CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25− CD45RBlow or CD45RBhigh CD4 T cells. We conclude that CD25+ CD45RBlow CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.

Lymphoid Potential, Probed before Circulation in Mouse, Is Restricted to Caudal Intraembryonic Splanchnopleura

Emergence of hemopoietic stem cells in the mammalian embryo has yet to be definitively allocated. Previously, we detected multipotent hemopoietic precursors in the region surrounding the dorsal aorta (paraaortic splanchnopleura) beginning at 8.5 days postcoitum (dpc). However, as circulation is already established, it remained unclear whether hemopoietic precursors arise in situ or are blood-delivered. By adding an organotypic step to our former culture system, we now detect lymphocyte and multipotent myeloid precursors from the intraembryonic splanchnopleura as early as 7.5 dpc. Under identical conditions, yolk sacs from the same embryos are unable to generate lymphoid progeny and have a reduced potential for myeloid differentiation and maintenance. Thus, if isolated before circulation, the yolk sac does not produce multipotent precursors and therefore does not contribute to definitive hemopoiesis in the mouse.

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7Rα) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11bloCD16−CD69hiLy49lo) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56hiCD16− NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

Identification of committed NK cell progenitors in adult murine bone marrow

Natural killer (NK) cells play important roles in innate immunity by lysing tumor and virally infected cells and by producing cytokines including interferon‐γ. While NK cell progenitors have been described in the fetal thymus, NK cell generation from hematopoietic stem cells (HSC) in the bone marrow (BM) occurs throughout life, and in athymic mice and humans. Interleukin (IL)‐15 promotes NK development in vitro and is essential for the generation of normal numbers of NK cells in vivo. By characterizing BM cells expressing IL‐15 receptor components, we found marked heterogeneity within the IL‐2 receptor β chain+ (CD122+) subset, which included cells uniquely committed to the NK lineage. These CD122+ NK cell precursors (NKP) are negative for markers used to identify mature NK cells, including NK1.1, DX5 and members of Ly‐49 family, and fail to demonstrate natural cytotoxicity against susceptible targetcells. In vitro culture of NKP generates mature lytic NK1.1+ cells at high frequencies, while they do not give rise to T, B, myeloid or erythroid cells under appropriate conditions. NKP lack transcripts associated with early B and T cell differentiation (pTα, λ5 and CD3ϵ), but express a group of genes (IL‐15Rα, Id2, GATA‐3 and Ets‐1) and the 2B4 marker, which may define NK cell committment. We propose that NKP represent the earliest adult BM precursor uniquely restricted to the NK cell lineage.

Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution

The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.

CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.

CX3CR1 expression is associated with the commitment of CSF-1R+ myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R+ CX3CR1+ macrophage/DC precursor (MDP) with other DC precursors and the role of CX3CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1+ inflammatory monocytes that differentiate into TipDCs during infection. CX3CR1 deficiency selectively impairs the recruitment of blood Gr1+ monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

The hare and the tortoise: an embryonic haematopoietic race

Cellular and gene therapy are obvious approaches to correct rare genetic disorders and, in the future, degenerative conditions. Both methods rely on the ability to identify and genetically modify stem cells in vitro, and on the reproducibility of in vivo colonization by the manipulated cells. The basic processes that are involved in the generation of haematopoietic cells in the embryo have been described in the past ten years, as a result of interdisciplinary approaches. These efforts have led to the identification of two independently generated types of haematopoietic progenitor cell, which differ in their potential for self-renewal and differentiation. These two populations might become key tools to understand the properties of stem cells.