Canonical Wnt signaling negatively modulates regulatory T cell function

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 Tcell 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 invitro and invivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector Tcells 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

Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation

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 (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a-/- HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis

The Nuclear Effector of Wnt-Signaling, Tcf1, Functions as a T-Cell-Specific Tumor Suppressor for Development of Lymphomas

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus

Mechanism of lymphomagenesis caused by Tcf1 deficiency.

<p>In the absence of Tcf1, thymocyte development is blocked at several stages (DN1, DN3, and ISP). Arrested thymocytes lack all isoforms of Tcf1, including the repressive isoforms. Loss of these repressive isoforms results in an up-regulation of Lef1, of which the long isoforms are most abundant in the thymus. Subsequently, Lef1 is capable of interacting with β-catenin as a compensatory mechanism, inducing deregulated Wnt signaling as measured by the high expression of <i>Axin2</i> in the vast majority of the lymphomas, forming a pre-leukemic stage. After additional mutations are acquired, of which activating mutation in <i>Notch1</i> frequently occur, and also other oncogenes such as <i>Mef2C</i> and <i>Lmo2</i> are likely candidates to be affected, full-blown lymphoma/leukemia develops.</p

Blocked Tcf1^−/− thymocytes show high Wnt-signaling in lymphoma development.

<p>(A) Tcf1^−/− mice were crossed to Conductin(Axin2)-LacZ reporter mice and sacrificed at different ages. Dot plots of four representative mice are shown: one control Tcf1^+/− LacZ (age 20 wk), one control Tcf1^−/− LacZ (age 9 wk), and two Tcf1^−/− Axin2-LacZ mice (age 20 wk, Tumor 1+ Tumor 2). Organs were collected and thymocytes were stained for CD4, CD8, CD3, CD44, and CD25 together with FDG to demonstrate Wnt reporter activity in the different thymocyte subsets. The corresponding thymus sizes for the four mice shown are 110×10⁶, 17×10⁶, 39×10⁶, and 175×10⁶ cells, respectively. Expression of Wnt reporter activity is shown per thymocyte subset, DP, ISP, and DN3 for control cells (filled), Tcf1^−/− cells (thin line), and Tcf1^−/− tumor cells (thick line). (B) RNA was isolated of total thymus and the expression level of Lef1 and Hes1 relative to Abl is shown for the control thymus and the two tumor samples as shown in (A). (C) The mean expression levels of Hes1 relative to Abl are shown for small tumors (<i>n</i> = 5) and large tumors (<i>n</i> = 25).</p

Tcf1^−/− lymphomas show deregulated Wnt signaling.

<p>(A) RNA isolated from thymi of 17 different mice was used for microarray analysis. Expression of Lef1, Tcf7, Myc, and Hes1 in the Tcf1^−/− mice without lymphoma (<i>n</i> = 4), Tcf1^+/− mice (control, <i>n</i> = 5) and Tcf1^−/− mice with a lymphoma (Lymphoma, <i>n</i> = 8) is shown. For the Tcf1^−/− lymphoma mice, the phenotype of the tumor is indicated on the horizontal axis. Columns represent independent RNA preparations of the different mice groups. A principal component analysis was performed using Wnt and Notch target genes. A PCA analysis shows clustering of the three groups as well as the effect each of the target genes has on the separation of these groups with samples of Tcf1^+/−, Tcf1^−/−, and Tcf1^−/− with tumors indicated by red, black, and green spheres, respectively. (B) Expression levels of Tcf1, Lef1, Axin2, Cyclin D1, cMyc, Hes1, and Deltex1 as determined by Affymetrix microarray were summarized and normalized using RMA, and the expression relative to Abl was plotted for each sample. Statistical significant differences (<i>p</i><0.05) as determined by Mann–Whitney U test are indicated by an asterisk. (C) A panel of 40 Tcf1^−/− thymic lymphomas and four control thymi (Tcf1^+/−) were analyzed by RQ-PCR. Expression data for Lef1 long (containing the β-catenin interacting domain), <i>Axin2</i>, <i>Deltex1</i>, and <i>Hes1</i> are shown relative to the house keeping gene <i>Abl</i>. Mann–Whitney U tests were performed to calculate the indicated <i>p</i> values.</p