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