To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells IntroductionHematopoietic stem cells (HSCs) are responsible for the continuous production of blood cells and consequently help to sustain immune function. This is achieved by their unique capacity to self-renew and ability to differentiate into all blood lineages. Several studies have implicated the Wnt-signaling pathway in the regulation of these processes, but its exact role is still not completely understood. 1,2 Upon binding of a Wnt protein to a Frizzled receptor and to a LRP5/6 coreceptor, an elaborate signaling route leads to cytoplasmatic accumulation and subsequent nuclear translocation of ␤-catenin, the key mediator of the Wnt-signaling pathway. In the absence of a Wnt protein, the levels of ␤-catenin are kept very low by the action of the so-called destruction complex consisting of the casein kinase I (CKI) and glycogen synthase kinase 3␤ (GSK-3␤) serine/threonine kinases, the tumor suppressor protein adenomatous polyposis coli (APC) and the scaffolding protein Axin. Phosphorylation of ␤-catenin by CKI and GSK-3␤ leads to its ubiquitination and subsequent breakdown in the proteossome. Activation of the Wnt pathway by a Wnt ligand results in inactivation of GSK-3␤ and consequent translocation of ␤-catenin to the nucleus. In the nucleus, ␤-catenin binds to members of the Tcf/Lef transcription factors family, thereby converting these proteins from transcriptional repressors into transcriptional activators. 3 The first evidence for a role of Wnt proteins in hematopoiesis was reported in studies showing that stromal cell lines transduced with Wnt1, Wnt5a, and Wnt10b have an in vitro stimulatory effect on mouse 4 and human 5 hematopoietic progenitors.Using Tcf1/Lef-GFP reporter assays, Wnt signaling was shown to be active in the highly HSCs enriched Lin Ϫ Sca1 ϩ c-Kit ϩ (LSK) population, both in vivo as well as in vitro after stimulation with purified Wnt3a. 6 Furthermore, Wnt3a treatment in vitro resulted in increased proliferation of LSK cells along with the maintenance of an immature phenotype and led to increased self-renewal as determined by transplantation assays. 7 Retroviral expression of a constitutively active form of ␤-catenin in Bcl2-transgenic LSK cells resulted in augmented multilineage repopulation capacity. In agreement, ectopic expression of the Wnt-signaling inhibitor Axin yielded opposite results. 6 However, subsequent gain-and loss-offunction approaches to fur...

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining ␤-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., ␤-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.double negative cells ͉ T cell development ͉ ␤-catenin ͉ Dickkopf

Our data indicate that the first 6 months of life represent a critical time window for the initiation of immunological changes resulting in the development of atopy. The selective development of a Th2 cytokine profile in high-risk children who develop atopy is due to increased production of Th2 cytokines, possibly caused by impaired allergen-induced IFN-gamma production in the neonatal period. Furthermore, the decreased allergen-induced IL-10 levels observed in the atopic children at 12 months of age may result in a lack of down-regulation of the inflammatory process.

It is a longstanding question which bone marrow-derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34 ؉ CD1a ؊ and CD34 ؉ CD1a ؉ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34 ؉ CD1a ؊ thymocytes but not in CD34 ؉ CD1a ؉ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34 ؉ CD1a ؊ subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34 ؉ CD1a ؊ thymocytes toward the T-cell lineage, as shown by T-cell receptor ␦ gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34 ؉ CD1a ؉ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34 ؉ CD1a ؊ and CD34 ؉ CD1a ؉ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity. IntroductionThe thymic microenvironment is exceptional in its ability to sustain production of T cells. 1 However, hematopoietic stem cells (HSCs) that will eventually give rise to T cells are derived from the bone marrow (BM). The nature of the thymus-seeding cell and its relation to several BM progenitors has remained elusive, despite being the subject of intense investigation. Furthermore, it is controversial whether cells commit to the T-cell lineage prethymically or intrathymically. 2,3 Aided by modern cell-sorting techniques, many studies in the mouse have recently readdressed these issues. Adult murine BM has been demonstrated to contain precursors with a restricted T-/B-lymphoid potential, so-called common lymphoid progenitors (CLPs). 2,4 However, the earliest thymic immigrants were shown to differ from CLPs in several aspects. 5 In peripheral blood, T-lineage potential appeared to be restricted to Lin Ϫ Sca-1 ϩ c-Kit ϩ (LSK) progenitor populations, rather than to CLPs. 6 Recently, two detailed analyses of the earliest subpopulations in the murine thymus showed variable lineage potential of different subsets. 7,8 Together, these studies point toward a model in which a range of BM-derived progenitors colonize the thymus, probably including multipotent progenitors and more lineage-restricted precursor cells. 9 For the human system, comparable experimental data are lacking. Determining lineage potential of early thymocytes will help to unveil the identity of the thymus-seeding cell in humans.In both humans and mice, the most immature ce...

The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor β-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/β-catenin/TCF pathway in the most immature (CD4−CD8−CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (∼30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/β-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool.

Tcf1 is known to function as a transcriptional activator of Wnt-induced proliferation during T cell development in the thymus. Evidence for an additional contrasting role for Tcf1 as a T-cell specific tumor suppressor gene is now presented.