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A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Thymocyte development is a complex process that requires precise regulation of differentiation and proliferation. Basic helix-loop-helix (bHLH) transcription factors have been shown to be crucial for proper T cell development. HEB and E2A are structurally and functionally related E proteins of the bHLH family. These proteins directly regulate the expression of a number of genes essential for lymphocyte development in a lineage- and stage-specific manner. Abrogation or compromise of their function results in the manifestation of B and T cell developmental defects. Genetic and biochemical studies have provided evidence of a significant degree of functional redundancy among E proteins. The existence of compensational abilities among different E proteins has hampered the investigation and elucidation of E protein function. As such, single gene knockouts demonstrate only limited defects in lymphocyte development. Double E2A-HEB knockouts that could eliminate E protein redundancy are embryonic lethal. In addition, conventional gene knockouts are not well-suited for discerning between intrinsic and extrinsic defects caused by E protein disruption. To eliminate functional compensation and to test the T cell intrinsic roles of E proteins during thymocyte development, we developed a conditional HEB-E2A double knockout. Specifically, we employed a loxP/Lck-Cre recombinase system to drive E protein deletion during early thymocyte development. Using this approach, we were able to reveal overlapping roles for HEB and E2A in thymocyte development that had been obscured in previous single gene knockout studies. We find that simultaneous deletion of HEB and E2A results in a severe block in thymocyte development at the DN to DP stage transition. This developmental block is accompanied by a dramatic decrease in total thymic cellularity, an increase in apoptosis, and a reduction of pTα expression. These developmentally arrested thymocytes exhibit increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7 signaling. Our findings suggest that E2A and HEB are not only critical for the regulation of T cell differentiation but are also necessary to retain developing thymocytes in cell cycle arrest prior to pre-TCR expression. Together, these results imply that E proteins are required to coordinate thymocyte differentiation and proliferation. / Dissertation

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.