Genes and signals controlling mammalian hematopoiesis.

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

Our current research is focused in four main areas: 1) characterization of the role of T cell antigen receptor (TCR) signals in T cell development. 2) identification and analysis of signal 'tuning' molecules that function downstream of the TCR that augment or inhibit TCR signaling. 3) Identification and characterization of previously undefined proteins that have important roles in T cell development. 4) Studies of the genes controlling the maintenance and self-renewal of Hematopoietic Stem Cells (HSCs) that may also have roles in stem cell like T cell tumors (T-ALL). Examining the role of T cell antigen receptor (TCR) signaling in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four different subunits of the multimeric TCR complex (CD3-gamma, -delta, -epsilon, -zeta). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated CD3-zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies indicated that TCR-ITAMs are functionally analogous but appear to act in concert to amplify TCR signals. In current studies, we are using conditional gene inactivation systems to more precisely analyze the importance of ITAM signaling at specific stages of development. We generated a "knock-in" model where mice express wild-type CD3-zeta but can be induced to express signaling incompetent (ITAM mutant) CD3-zeta by Cre-recombinase. Using this model, we made the surprising discovery that the CD3-zeta ITAMs can perform both activating/amplifying and inhibitory roles in TCR signaling depending upon the affinity of the TCR/ligand interaction. We found that CD3-zeta ITAMs regulate ligand discrimination by inhibiting signaling by low affinity TCR/ligand interactions and by contributing to activation by high affinity TCR/ligand interactions. In current studies, we are seeking to leverage this discovery to enhance the activity of T cells that express low affinity tumor specific TCRs for cancer treatment in humans. Identifying molecules that 'fine-tune' the TCR signal. Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR ITAMs. An initial search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a static inhibitory co-receptor, CD5 regulation by TCR signaling provides a feedback mechanism to 'fine-tune' the overall TCR signaling response during thymocyte selection since the expression of CD5 depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling threshold 'window' of positive selection. Since little is known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. We have also begun a search for additional tuning molecules using microarray and RNA-Seq screens and literature searches. We believe that the identification of such molecules will be relevant to the diagnosis and treatment of human autoimmune diseases and cancer (similar to checkpoint inhibitors) since they function to set the activation threshold for naive T cells. In current studies we are testing if conditional deletion of CD5 in T cells enhances their tumor reactivity in the mouse model. Identification of new genes important for T cell development. Themis: We identified a novel T-lineage restricted protein designated Themis and generated germline and conditional Themis deficient mice. Themis is the prototype of a new family of proteins in metazoans that are defined by the presence of a novel domain called the CABIT module. We discovered that CABIT modules function by binding to tyrosine phosphatase catalytic domains and inhibit catalytic activity. By inhibiting the tyrosine phosphatase SHP-1 Themis enhances TCR signaling in developing thymocytes enabling them to successfully undergo positive selection mediated by low affinity self-peptides. FBXL12: We recently identified and characterized a new T cell protein called Fbxl12. We found that the Fbxl12 functions as a subunit of an SCF ubiquitin ligase complex that degrades the cell cycle inhibitor Cdkn1b resulting in proliferation. Previous data had shown that a related protein, Fbxl1 also targets Cdkn1b for degradation in thymocytes. We investigated the function of Fbxl12 by generating conditional knockout mice, comparing the phenotype to Fbxl1 knockout mice, and examining the phenotype of Fbxl1/Fbxl12 double knockout mice. We found that both Fbxl1 and Fbxl12 are required for proliferation of thymocytes at a stage called beta-selection which is controlled by concurrent signals transduced by the pre-TCR and Notch. Our results demonstrated that Fbxl1 expression is induced by Notch whereas Fbxl12 expression is induced by the pre-TCR and that Fbxl1 and Fbxl12 function additively to degrade Cdkn1b. This mechanism explains the requirement for simultaneous Notch and pre-TCR signaling for beta-selection associated proliferation. Role of Ldb1 in hematopoiesis. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to loss of self-renewal capability and the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. Several years ago we initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis as prior work had suggested a function for Ldb1 in the hematopoietic system. These experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggested that Ldb1-nucleated multi-subunit transcription complexes control HSC maintenance. We also found that another Ldb1 complex with different subunits controls the expression of erythroid genes demonstrating that Ldb1 functions as a key factor in modular transcription complexes that regulate multiple gene programs. Role of Ldb1 complexes in T-ALL. Our current studies are focused on exploring the role of Ldb1 complexes in the maintenance/self-renewal of immature thymocytes that resemble HSCs. Our results suggest that abnormal self-renewal of these cells predisposes to T-Cell Acute Lymphoblastic Leukemia (T-ALL). Using a model of T-ALL that resembles an aggressive form of Early T Progenitor human T-ALL (ETP T-ALL) we found that Ldb1 is required for abnormal self-renewal of thymocytes prior to oncogenesis and is required for the induction of ETP T-ALL in mice. These results identify Ldb1 complexes as potential targets for treatment of human T-ALL.