The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

Em estudos anteriores desenvolvidos por este grupo de pesquisa uma expressão mais elevada de alvos transcricionais e componentes da via NF-kB, bem como altos níveis de NOTCH1, foi identificada em células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) quando comparadas às CTH CD34+ de medula óssea (MO). Este grupo verificou ainda, por comparação das células CD34+ com as CD133+ (mais primitivas) que diversos fatores de transcrição (FT) envolvidos com o potencial de hemangioblasto, com a autorenovação das CTH, e com a diferenciação linfóide; como: RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 e HOXB4 apresentaram-se mais expressos em células mais primitivas. A potencial participação das vias Notch e NF-kB na regulação destes FT tem importância conceitual e prática no entendimento da biologia das CTH, e dos processos envolvidos na diferenciação destas células. Com isto em vista, este projeto teve como objetivo, estudar o papel da via NF-kB e da via Notch na regulação destes FT. Para isso, um modelo experimental in vitro, de diferenciação de CTH CD34+ em linfócitos T, foi utilizado e a influência de fatores agonistas e inibidores farmacológicos destas vias, foram avaliados por citometria de fluxo e PCR em tempo real. Nossos resultados evidenciam o papel da via Notch na regulação transcricional de HOXB4 e GATA3 em células-tronco hematopoéticas CD34+ humanas, o que foi confirmado com base na expressão dos alvos diretos de Notch (HEY1 e HES1). Notamos ainda, que a expressão dos transcritos HES1, GATA3 e HOXB4 é prejudicada pela síntese protéica das CTH, uma vez que quando empregamos o prétratamento com a droga CHX há aumento da transcrição dos mesmos. Também podemos inferir que a ação do TNF- é positiva sobre esses transcritos, já que quando o utilizamos há elevação do nível de expressão desses transcritos, com exceção a HES1. Em relação ao cocultivo das CTH com as células estromais de camundongos, verificamos que apenas a linhagem OP9-DL1 detém a capacidade de promover a diferenciação celular T, e isso foi comprovado pelo surgimento de células comprometidas com a linhagem linfocítica T, através da presença dos marcadores de superfície específico CD7+ e CD1a+. Esses resultados auxiliarão na compreensão dos mecanismos moleculares de regulação transcricional envolvidos não apenas na diferenciação de linfócitos T, mas também na manutenção de um estado mais primitivo das CTH. Este conhecimento pode vir a contribuir com o desenvolvimento ou otimização de protocolos laboratoriais visando à expansão de CTH ou geração de células T para usos terapêuticos. / In previous studies by this research group a higher expression of transcriptional targets and components via NF-kB, as well as high levels of NOTCH1, was identified in hematopoietic stem cells (HSC) CD34 + cells from umbilical cord blood (UCB) compared to CD34 + hematopoietic stem cells from bone marrow (BM). This group also found, by comparing the CD34 + cells with CD133 + (more primitive) that several transcription factors (TF) involved in the potential of hemangioblast, with self-renewal of hematopoietic stem cells and to differentiated lymphocytic; as Runx1 / AML1, GATA3, USF1, TAL1/SCL, HOXB4 and HOXA9 were more expressed in more primitive cells. The potential involvement of Notch signaling pathways and NF-kB in the regulation of FT has conceptual and practical importance in understanding the biology of HSC, and the processes involved in differentiation of these cells. With this in mind, this project aimed to study the role of NF-kB pathway and Notch signaling in the regulation of FT. For this, an experimental model in vitro differentiation of CD34 + hematopoietic stem cells into T lymphocytes, was used and the influence of pharmacological agonists and inhibitors of these pathways were evaluated by flow cytometry and real-time PCR. Our results highlight the role of Notch signaling in the transcriptional regulation of GATA3 and HOXB4 in hematopoietic stem cells CD34 + human, which was confirmed based on the expression of direct targets of Notch (HES1 and HEY1). We also note that the expression of transcripts HES1, GATA3 and HOXB4 protein synthesis is hampered by the HSC, since when we use the pre-treatment with the drug there CHX increased transcription thereof. We can also infer that the action of TNF- is positive about these transcripts, since when we use it for raising the level of expression of these transcripts, except the HES1. In relation to the HSC coculture with stromal cells of mice, we found that only the line-DL1 Op9 has the ability to promote T cell differentiation, and this was evidenced by the appearance of cells committed to the T lymphocyte lineage, through the presence of specific surface markers CD7 + and CD1a +. These results will help understand the molecular mechanisms of transcriptional regulation involved not only in the differentiation of T lymphocytes, but also in maintaining a more primitive state of HSC. This knowledge may contribute to the development or optimization of laboratory protocols aimed at the expansion of HSC or generation of T cells for therapeutic use.

Em estudos anteriores desenvolvidos por este grupo de pesquisa uma expressão mais elevada de alvos transcricionais e componentes da via NF-kB, bem como altos níveis de NOTCH1, foi identificada em células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) quando comparadas às CTH CD34+ de medula óssea (MO). Este grupo verificou ainda, por comparação das células CD34+ com as CD133+ (mais primitivas) que diversos fatores de transcrição (FT) envolvidos com o potencial de hemangioblasto, com a autorenovação das CTH, e com a diferenciação linfóide; como: RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 e HOXB4 apresentaram-se mais expressos em células mais primitivas. A potencial participação das vias Notch e NF-kB na regulação destes FT tem importância conceitual e prática no entendimento da biologia das CTH, e dos processos envolvidos na diferenciação destas células. Com isto em vista, este projeto teve como objetivo, estudar o papel da via NF-kB e da via Notch na regulação destes FT. Para isso, um modelo experimental in vitro, de diferenciação de CTH CD34+ em linfócitos T, foi utilizado e a influência de fatores agonistas e inibidores farmacológicos destas vias, foram avaliados por citometria de fluxo e PCR em tempo real. Nossos resultados evidenciam o papel da via Notch na regulação transcricional de HOXB4 e GATA3 em células-tronco hematopoéticas CD34+ humanas, o que foi confirmado com base na expressão dos alvos diretos de Notch (HEY1 e HES1). Notamos ainda, que a expressão dos transcritos HES1, GATA3 e HOXB4 é prejudicada pela síntese protéica das CTH, uma vez que quando empregamos o prétratamento com a droga CHX há aumento da transcrição dos mesmos. Também podemos inferir que a ação do TNF- é positiva sobre esses transcritos, já que quando o utilizamos há elevação do nível de expressão desses transcritos, com exceção a HES1. Em relação ao cocultivo das CTH com as células estromais de camundongos, verificamos que apenas a linhagem OP9-DL1 detém a capacidade de promover a diferenciação celular T, e isso foi comprovado pelo surgimento de células comprometidas com a linhagem linfocítica T, através da presença dos marcadores de superfície específico CD7+ e CD1a+. Esses resultados auxiliarão na compreensão dos mecanismos moleculares de regulação transcricional envolvidos não apenas na diferenciação de linfócitos T, mas também na manutenção de um estado mais primitivo das CTH. Este conhecimento pode vir a contribuir com o desenvolvimento ou otimização de protocolos laboratoriais visando à expansão de CTH ou geração de células T para usos terapêuticos. / In previous studies by this research group a higher expression of transcriptional targets and components via NF-kB, as well as high levels of NOTCH1, was identified in hematopoietic stem cells (HSC) CD34 + cells from umbilical cord blood (UCB) compared to CD34 + hematopoietic stem cells from bone marrow (BM). This group also found, by comparing the CD34 + cells with CD133 + (more primitive) that several transcription factors (TF) involved in the potential of hemangioblast, with self-renewal of hematopoietic stem cells and to differentiated lymphocytic; as Runx1 / AML1, GATA3, USF1, TAL1/SCL, HOXB4 and HOXA9 were more expressed in more primitive cells. The potential involvement of Notch signaling pathways and NF-kB in the regulation of FT has conceptual and practical importance in understanding the biology of HSC, and the processes involved in differentiation of these cells. With this in mind, this project aimed to study the role of NF-kB pathway and Notch signaling in the regulation of FT. For this, an experimental model in vitro differentiation of CD34 + hematopoietic stem cells into T lymphocytes, was used and the influence of pharmacological agonists and inhibitors of these pathways were evaluated by flow cytometry and real-time PCR. Our results highlight the role of Notch signaling in the transcriptional regulation of GATA3 and HOXB4 in hematopoietic stem cells CD34 + human, which was confirmed based on the expression of direct targets of Notch (HES1 and HEY1). We also note that the expression of transcripts HES1, GATA3 and HOXB4 protein synthesis is hampered by the HSC, since when we use the pre-treatment with the drug there CHX increased transcription thereof. We can also infer that the action of TNF- is positive about these transcripts, since when we use it for raising the level of expression of these transcripts, except the HES1. In relation to the HSC coculture with stromal cells of mice, we found that only the line-DL1 Op9 has the ability to promote T cell differentiation, and this was evidenced by the appearance of cells committed to the T lymphocyte lineage, through the presence of specific surface markers CD7 + and CD1a +. These results will help understand the molecular mechanisms of transcriptional regulation involved not only in the differentiation of T lymphocytes, but also in maintaining a more primitive state of HSC. This knowledge may contribute to the development or optimization of laboratory protocols aimed at the expansion of HSC or generation of T cells for therapeutic use.