Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.

Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(-/)- mice.

Interleukin 7 is a crucial factor for the development of murine T and B lymphocytes. We now report that, in the absence of interleukin 7, B lymphocyte production takes place exclusively during fetal and perinatal life, ceasing after 7 wk of age. In peripheral organs, however, the pool of B lymphocytes is stable throughout adult life and consists only of cells that belong to the B1 and marginal zone (MZ) compartments. This is accompanied by a 50-fold increase in the frequency of immunoglobulin (Ig)M- and IgG-secreting cells, and the concentration of serum immunoglobulins is increased three- to fivefold. Both the MZ phenotype and the increase in serum IgM are T cell independent. These findings reveal a previously undescribed pathway of B lymphopoiesis that is active in early life and is interleukin 7 independent. This pathway generates B1 cells and a normal sized MZ B lymphocyte compartment.

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.

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-gamma. 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 beta 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 target cells. 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 (pTalpha, lambda5 and CD3epsilon), but express a group of genes (IL-15Ralpha, Id2, GATA-3 and Ets-1) and the 2B4 marker, which may define NK cell commitment. We propose that NKP represent the earliest adult BM precursor uniquely restricted to the NK cell lineage.

Inhibition of Plasmodium yoelii blood-stage malaria by interferon alpha through the inhibition of the production of its target cell, the reticulocyte.

The effect of a recombinant hybrid human interferon alpha (IFN-alpha) (which cross-reacts with murine cells) on C57BL/6 mice infected with Plasmodium yoelii sporozoites or parasitized erythrocytes was determined. IFN-alpha did not inhibit the development of the parasite in the liver, but it did reduce the blood parasite load and the hepatosplenomegaly induced by the infection in mice injected with blood-stage parasites. The extent of anemia in IFN-alpha-treated and control mice was similar, despite the lower parasite load in the IFN-alpha-treated mice. The reduced blood parasite load in IFN-alpha-treated mice was associated with reduced erythropoiesis and reticulocytosis. As reticulocytes are the preferred target cells for the strain of P yoelii used (P yoelii yoelii 265 BY), it was postulated that the inhibition of reticulocytosis in IFN-alpha-treated mice was causally related to the observed decreased blood parasite load. This was supported by the finding that IFN-alpha inhibited a different strain of P yoelii (17X clone A), which also displays a tropism for reticulocytes, but not a line of Plasmodium vinckei petteri, which infects only mature red blood cells. As human malaria species also display different tropism for reticulocytes, these findings could be relevant for people coinfected with multiple Plasmodium species or strains or coinfected with Plasmodium and virus. (Blood. 2001;97:3966-3971)

Mice triallelic for the Ig heavy chain locus: implications for VHDJH recombination.

V(H)DJ(H) recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of V(H)DJ(H) recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2(-/-) mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional mu-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.

Pluripotent hematopoietic stem cell development during embryogenesis.

Significant progress has been made towards a better understanding of the establishment of hematopoiesis in the embryo. Hematopoietic precursors have been shown to arise independently in the yolk sac and in the intra-embryonic mesoderm. From the combined analysis of differentiation potentials, expression patterns and mutant phenotypes, a picture has emerged: definitive hematopoietic precursors are transiently generated in a specific environment close to the endothelium of the main arteries.

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 (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) 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(+) CD45RB(low) 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 CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) 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.

Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality.

Polymerase zeta (Pol zeta) is an error-prone DNA polymerase [1], which in yeast is involved in trans-lesion synthesis (TLS) and is responsible for most of the ultraviolet (UV) radiation-induced and spontaneous mutagenesis [2-4]. Pol zeta consists of three subunits: REV1, a deoxycytidyl-transferase [5]; REV7, of unclear function [6]; and REV3, the catalytic subunit. REV3 alone is sufficient to carry out TLS, but association with REV1 and REV7 enhances its activity [5, 7]. Experiments using human cells treated with UV radiation indicate also that mammalian Pol zeta is involved in TLS [7]. The peculiar mutagenic activity of Pol zeta [4,7,8] suggests a possible role in somatic hypermutation of immunoglobulin (Ig) genes [9]. Here, we report that, unlike in yeast where the REV3 gene is not essential for life [4], disruption of the mouse homologue (Rev3l) resulted in early embryonic lethality. In Rev3l(-/-) embryos, no haematopoietic cells other than erythrocytes could be identified in the yolk sac. Rev3l(-/-) haematopoietic precursors were unable to expand in vitro and no haematopoietic cells could be derived from the intraembryonic haematogenic compartment (splanchnopleura). Fibroblasts could not be derived from the Rev3l(-/-) embryos, and Rev3l(-/-) embryonic stem (ES) cells could not be obtained. This is the first evidence that an enzyme involved in TLS is critical for mammalian development.

Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation.

In the mouse, the number and the differentiation potential of thymic migrants remain controversial. A fetal thymic organ culture under limiting dilution conditions allowed us to show a 130-fold increase in the numbers of T cell precursors in the embryonic thymus between days 12 and 14 of gestation. A comparative analysis of the most immature thymocytes at these two stages revealed that: (1) CD44(+)CD25(-) (DN1) thymocytes at 14 days post coitum (dpc) efficiently differentiate into mature T cells both in vivo and in vitro; (2) 12dpc thymocytes exhibit a low frequency of T cell precursors and were unable to generate a detectable progeny after in vivo intrathymic transfer. A 48-h organ culture of 12dpc thymic lobes did neither correct the low frequency of T cell precursors nor the absence of expression of T cell-specific genes observed in 12dpc thymocytes. We thus concluded that a fraction of recent thymic immigrants contribute to the observed properties in DN1 14dpc thymocytes. We show that increasing numbers of T cell precursors migrate to the thymus from 11 to 14 dpc. We propose that the first thymic immigrants do not contribute significantly to T cell generation which depends on the subsequent colonization by cells with a high T cell precursor potential.

Evidence for two subgroups of CD4-CD8- NKT cells with distinct TCR alpha beta repertoires and differential distribution in lymphoid tissues.

NKT cells are a subset of T lymphocytes that is mainly restricted by the nonclassical MHC class I molecule, CD1d, and that includes several subpopulations, in particular CD4+ and CD4-CD8- (DN) cells. In the mouse, differential distribution of these subpopulations as well as heterogeneity in the expression of various markers as a function of tissue localization have been reported. We have thus undertaken a detailed study of the DN NKT cell subpopulation. With a highly sensitive semiquantitative RT-PCR technique, its TCR repertoire was characterized in various tissues. We found that mouse DN NKT cells are a variable mixture of two subgroups, one bearing the invariant Valpha14 chain paired to rearranged Vbeta2, Vbeta7, Vbeta8.1, Vbeta8.2, or Vbeta8.3 beta-chains and the other exhibiting unskewed alpha- and beta-chains. The proportion of these subgroups varies from about 100:0 in thymus, 80:20 in liver, and 50:50 in spleen to 20:80% in bone marrow, respectively. Finally, further heterogeneity in the tissue-derived DN NKT cells was discovered by sequencing extensively Vbeta8.2-Jbeta2.5 rearrangements in individual mice. Despite a few recurrences in TCR sequences, we found that each population exhibits its own and broad TCRbeta diversity.

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.

Regulatory CD4 T cells control the size of the peripheral activated/memory CD4 T cell compartment.

The mechanisms leading to stable T cell numbers in the periphery of a healthy animal are, to date, not well understood. We followed the expansion of CD45RBhigh (naive) and CD45RBlow (activated/memory) CD4 T cells transferred from normal mice into syngeneic Rag-20/0 recipients and the dynamics of peripheral reconstitution when both populations were coinjected. Naive cells acquired an activated phenotype and showed a high proliferative capacity that was dependent on the environment in which the recipients were kept (specific pathogen-free vs conventional housing conditions), the age of the recipients, and the presence of CD45RBlow T cells in the injected cohort. CD45RBlow CD4 T cells protected the host from CD45RBhigh CD4 T cell-induced inflammatory bowel disease and showed a limited degree of expansion. CD45RBlow CD4 T cells isolated from GF mice also showed the ability to prevent inflammatory bowel disease, indicating that at least part of the natural regulatory T cells are self-reactive. The results indicate that 1) peripheral T cell expansion in lymphocyte-deficient recipients represent classical immune responses, which are mainly promoted by exogenous Ags and 2) natural regulatory T cells control the size of the activated/memory peripheral CD4 T cell compartment.

The splanchnopleura/AGM region is the prime site for the generation of multipotent hemopoietic precursors, in the mouse embryo.

The first hemopoietic cells were found in the yolk sac of mammalian embryos. Based on this observation it was postulated that hemopoietic stem cells are generated in this location. In the last few years, however, increasing evidence indicates that there is an independent site of hemopoietic cell generation, in the embryo proper, designated the paraaortic splanchnopleura/aorta, gonad, mesonephros region. Precursors of hemopoietic cells are found in this region before circulation is established between the yolk sac and the embryo proper. In contrast to the hemopoietic cells found in the yolk sac, the ones found in the embryo proper are multipotent lympho-myeloid precursors that rapidly acquire the capacity to reconstitute the hemopoietic system of adult irradiated recipients. We propose that these are the founder cells of adult definitive hematopoiesis.

Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice.

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.

Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites.

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/ aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

Critical involvement of Tcf-1 in expansion of thymocytes.

T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4- 8- thymocytes are observed at two distinct stages (CD44+ 25+ and CD44- 25-), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1(-/-) progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1(-/-) mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/beta-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.

Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis.

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.

Extensive junctional diversity of Ig heavy chain rearrangements generated in the progeny of single fetal multipotent hematopoietic cells in the absence of selection.

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.