Signaling During Thymic Selection

  • Thymic selection ensures the development of T-cells that are tolerant to self-antigens yet display a wide range of specificities. Even minor dysregulation in this process can lead to serious autoimmune and inflammatory diseases. Double positive (DP) thymocytes expressing T-cell receptors (TCRs) that recognize MHC, with moderate affinity are positively selected and form labile immune synapses. Most high affinity TCRs, which organize into stable synapses are deleted but some give rise to regulatory T-cells (nTreg) that suppress peripheral autoimmune responses. The molecular processes that determine if a thymocyte is deleted or becomes nTreg are not known. High affinity TCR interactions induce b-catenin/Tcf signaling by deubiquitinating and stabilizing b-catenin and this determines the outcome of thymic selection. TCR engagement in DP-cells expressing conditionally stabilized b-catenin instructs reduced generation of nTreg and increased negative-selection mediated by overexpression of Bim. Importantly these mice suffer frequent gut inflammation and dysplasia. In contrast ablation of the nuclear partner of b-catenin, Tcf-1, instructs rescue from negative-selection. Besides the role of b-catenin signaling in the fate of thymocytes indicated by these findings, this protein has an additional structural role in the immune synapse. Our hypothesis is that high affinity TCRs dictate cell fate decisions including tolerance because they transmit sufficient signals to form an immune synapse and thereby stabilize b-catenin, which is then available for activation of the b-catenin/Tcf signaling pathway. We are testing the hypothesis that β-catenin/Tcf signaling is a switch that regulates thymocyte fate decisionsby relating gradual increases in TCR signaling to the activation of b-catenin signaling and to the gene expression events that are enabled. These studies are also expected to establish, the role of β-catenin/Tcf signaling in the generation and function of nTreg. We are also testing the hypothesis that strong TCR signals mobilize b-catenin to the immune synapse and activate its deubiquitination. The recruitment and the role of b-catenin in the immune synapse is monitored in response to increasing TCR signals and the deubiquitinase(s) (DUBs) that target β-catenin in this process are been identified. These studies are expected to determine how thymocytes differentiate between positive and negative selection or Treg development. They will therefore provide a better understanding of the molecular predisposition to chronic inflammatory diseases, autoimmunity, and cancer. Furthermore this research is likely to reveal a functional link between the "adhesion" and the "transcription" pools of b-catenin, which will provide a fundamental paradigm shift with implications in multiple developmental processes and in the etiology of diseases that extend beyond the mechanisms of thymic selection. Several innovative approaches are been used for these studies including stringent systems for expression profiling, specialized imaging and advanced biochemistry. Furthermore novel in vivo models are been used including an inducible CD4CreEr transgene and strains to knockdown or over-express DUBs.


Mechanisms of Genomic Instability in T-cell Transformation

Genomic instability is the underlying cause of cancer initiation, progression, as well as resistance to therapy. While chromosomal aberrations are common and assumed to be causative in nearly all cancers they are best characterized in hematopoietic malignancies. Most T-cell leukemia/lymphoma have recurrent chromosomal translocations leading to oncogene activation. Lack of understanding of the molecular mechanism(s) that predispose to genomic instability hampers the development of effective cancer therapies. Here we propose to use novel genome wide approaches to dissect the molecular basis of genomic instability that will have ramifications for improved therapies of genomically unstable cancers.

Text Box:  Fig 1: t(14;15)(C2;D1) translocations and chromosome 15 amplifications in b-catenin leukemia. A. Representative metaphase of b-catenin lymphoma analyzed by SKY. Seen in 7 out of 8 lymphomas. Red boxes are aberrant chromosomes. B. Representative FISH analysis with the indicated BAC probes. C. Cartoon of the translocation. The location of BACs used is shown. Organization of the translocated loci in the wt chromosomes is indicated. D. Sequence of the chimeric Pvt1 Ca transcript and cartoon of the genomic organization.           

 We are comparing a model of genomically unstable T-cell lymphoma we have recently generated by conditionally activating b-catenin to relevant human T-cell lymphomas. Activation of Wnt signaling and mutations in b-catenin have been detected in T-cell leukemia/lymphoma. These genomically unstable malignancies have particularly bad prognosis4 with only 10% cure rate. Therapeutic progress is hindered by a complete lack of understanding their pathogenesis. Our model provides an invaluable tool for revealing the distinguishing properties of genomically unstable cancers. This knowledge can ultimately be used to improve cancer diagnosis and treatment. Specifically, we have demonstrated that conditional Cre mediated activation of b-catenin (CD4CreCtnnb1ex3) leads to T-cell lymphomas by promoting genomic instability as evidenced by the detection of recurrent TCRa/Myc translocations (Fig 1) that are Identical to translocations seen in human T-cell lymphomas leading to c-Myc activation that is a common outcome of translocations in this disease. Mouse lymphoma models that can activate b-catenin such as conditional ablation of PTEN, or expression of active AKT also have TCRa/Myc translocations and require b-catenin signaling. Together these observations place b-catenin in a central position for promoting genomic instability.

         Deregulation of b-catenin has been linked with genomically unstable cancers, however, the signals generating this instability have yet to be identified. We predict that activated b-catenin promotes instability by altering the expression and/or the chromatin state of key target genes. b-Catenin has been shown to regulate the expression of target genes by promoting trimethylation of histone3 at lysine 4 (H3K4me3) and at lysine 79 (H3K79me3). In addition it has been shown to control gene expression by competing with the histone deacetylace HDAC1. These epigenetic effects of b-catenin are relevant for genomic stability, because chromatin accessibility is intimately linked with DNA repair. Histone acetylation and chromatin remodeling have been established in yeast, as mechanisms to open the chromatin for the repair of DSBs and UV-induced lesions. Chromatin opening by histone acetylases (HATs) to repair DNA is followed by the recruitment of histone deacetylases (HDACs) near DSBs, which reduce histone acetylation and restore higher-order chromatin after repair is completed. This is essential for ensuring genome stability because it regulates the recruitment of checkpoint and DNA-damage response mediators that directly bind to open histone marks.