Genes and signals controlling mammalian hematopoiesis.

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Our 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 and that may represent targets for immunotherapy in humans. 3) Identification and characterization of new molecules 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 (zeta, CD3-gamma, -delta, -epsilon). 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 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 demonstrated that TCR-ITAMs are functionally analogous but appear to act in concert to amplify TCR signals. The full complement of TCR ITAMs was found to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). In current studies, we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development and after T cell development and thymocyte selection is finished. We generated a knock-in model where mice express wild-type zeta but can be induced to express signaling incompetent (ITAM mutant) zeta by Cre-recombinase. Using this model we can test the effect of reduced TCR signaling potential on TCR function at various stages of development or in particular T cell subsets. 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 FACS-based 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. The identification of such molecules may be relevant to the diagnosis and treatment of human autoimmune diseases and tumors (similar to checkpoint inhibitors) since these molecules function to set the activation threshold for naive T cells. Identification of new genes important for T cell development. Themis: We identified a novel T-lineage restricted protein designated Themis. Biochemical studies indicate that Themis functions in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling in thymocytes undergoing positive selection. Germline and and conditional Themis deficient mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and in thymocyte selection. 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 the tyrosine phosphatase SHP-1 and inhibiting SHP-1 catalytic activity. By inhibiting 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 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 is induced by Notch whereas Fbxl12 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 Hematopoietic Stem (HSC) cell maintenance and self-renewal. 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 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 suggest that Ldb1-nucleated multi-subunit transcription complexes may control HSC maintenance. Deletion of Ldb1 in HSCs resulted in loss of HSCs revealing that Ldb1 complexes function as 'master regulators' of the transcriptional program regulating HSC maintenance/self-renewal. Role of Ldb1 complexes in T-ALL. 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 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 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 targets for treatment of human T-ALL.