The thymus is the principal site of T-cell development and maturation. Failure to develop a functional thymus leads to severe immunodeficiency, while partially incorrect function of the organ can lead to a variety of autoimmune diseases as well as higher risk for infections and cancer. The thymus is organized into cortical and medullary regions, which are functionally distinct. The diverse array of thymic epithelial cells (TEC) are the key components of the thymic stroma, both the cortical and medullary TEC subsets are responsible for the establishment of a self-tolerant and self-restricted T-cell repertoire. The thymus is most active in young individuals, and undergoes a progressive naturally occurring involution from birth, which accelerates after puberty. Thymic involution is characterized by loss of thymus organization and function, including an overall reduction in the amount of functional thymic tissue. This results in decreased production of new naïve T-cells, and contributes to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Involution of the thymus, both natural and in response to different therapies such as chemotherapy, raises interest in developing cell based treatment methods that will allow the restoration of the thymic architecture and so elevate immune reconstitution in vivo. The cellular mechanisms by which the postnatal thymus is maintained during homeostasis and involution are currently unknown. The earliest thymic progenitors in the thymus express Plet1; it has been established that from E12.5 to E15.5 these cells when purified are able to generate all thymic epithelial cell types and initiate thymus organogenesis. However, at least the latter capacity is reported to be lost from E18.5. A number of papers published provide evidence for the existence of both bipotent and unipotent TEC progenitors in the adult thymus. However the identity of these cells remains unknown, nor has the relationship between the mature and immature postnatal TEC compartments been established. The aim of my research was to investigate the cellular mechanism(s) that maintain the postnatal thymus. Specifically, I aimed to determine whether the thymus is maintained by a stem cell mechanism or by division of terminally differentiated thymic epithelial cells, and whether or not postnatal thymic epithelial stem/progenitor cells express functionally relevant levels of the transcription factor Foxn1. To address these aims, I used two approaches: in vivo genetically heritable lineage tracing and a novel grafting assay to assess the contribution of different lineages of TEC. This thesis describes the characterization of a novel mouse strain, the Foxn1CreERt2 line, which was predicted to allow conditional inducible manipulation of gene expression in TEC. I show that this deletor strain, while thymic epithelial cellspecific, could induce cre-mediated recombination in only in a low proportion of TEC and thus could not be used to address the initial aim of this work as described above. However, lineage tracing experiments using this line have provided evidence for a persistent cortical thymic epithelial progenitor/stem cell type, that was capable of rapid expansion within the cortical compartment over time. In parallel with characterisation of the Foxn1CreERt2 strain, I investigated the potential of various defined epithelial populations to contribute to the thymic environment in an assay of TEC potency. Using this technique I have established the potential of defined TEC subpopulations isolated from postnatal mice to generate cortical and medullary TEC. Among the populations analysed I have identified a minor TEC subset that can robustly contribute to both cortical and medullary TEC that coexpress Ly51 and Plet1. I have further shown, using a limiting dilution approach, that this population contains a postnatal common thymic epithelial stem/progenitor cells, present at a frequency of between 87.5 and 92.5 within this population. I have also produced evidence of a unipotent cortical progenitor population that is capable of long term expansion in vivo.

The thymus is the primary lymphoid organ responsible for the development and maturation of T lymphocytes (aka T-cells) in vertebrates. The complex architecture of the thymic microenvironment orchestrates the formation of a diverse and self-tolerant T-cell repertoire capable of supporting the development and maintenance of a functional immune system. The main component of this microenvironment, the thymic epithelium, is crucially required to direct thymus organogenesis and homeostasis, and to mediate T-cell repertoire development and selection. The thymic epithelial progenitor cells (TEPCs) from which the mature thymus develops originate from the endoderm of the 3rd pharyngeal pouch by embryonic day 9 in mouse development (or early week 6 in human embryos). Expression of the transcription factor FOXN1 is required to drive TEPCs differentiation in each thymic epithelial lineage (TEC), while the absence of functional FOXN1 causes athymia. Moreover, forced expression of Foxn1 in mouse embryonic fibroblasts (MEFs) converts these MEFs into TECs that can support the development of a normal thymic system. Despite the great therapeutic potential that TEPCs present in regenerative medicine, there is currently no detailed model describing regulation of the TEPC state and its differentiation into cortical (c) and medullary (m) TECs, or explaining the dominant role of FOXN1 in the thymic epithelial system. Comparative transcriptomics analysis in conjunction with pathway enrichment analysis of the developing TEPCs could reveal the signalling pathways that regulate the early TEPC state and progression into differentiation. Additionally, integrative bioinformatics analysis of transcriptomics and genomics datasets could identify the functional networks that are directly regulated by FOXN1 during early TEC progression. In this thesis I provide, for the first time, an in silico model explaining fetal TEPC differentiation into the functionally distinct TEC lineages, in the cellular, molecular and signalling contexts of thymus early development. Furthermore, I present evidence which suggests that FOXN1 could be a pioneer factor, capable of fully establishing the transcriptional programme that underpins thymic epithelial cell identity and function. Finally, in this thesis, I introduce the development of an interactive thymic-specific database that provides a platform for easy access, analysis and integration of curated bioinformatics datasets.

Neuroendocrine cell populations were systematically studied and characterized in the thymus, an avian primary immune organ. The expression of the specific mRNAs for both Chromogranin A (CgA) and Carboxypeptidase E (CpE) in the thymus was first verified by RT-PCR. Additional evidence using immunofluorescent dual labeling, has demonstrated for the first time the co-existence of CgA and CpE in identical neuroendocrine cells at the protein level in a vetebrate primary lymphoid organ. These CpE- and CgA-positive cells were primarily found in the transition zone between the cortex and the medulla of the thymic lobules, an area known to contain numerous arterioles and to be heavily innervated by the autonomic nervous system, suggesting that these cell population can potentially receive input from each other, from the autonomous nervous system, from the circulation, or all of the above. (Neuro)endocrine messenger molecules produced by the thymic microenvironment, such as somatostatin (SST), seem to play a potentially important immunomodulatory role with regard to cell proliferation, differentiation, and migration, as well as cytokine production. The results showed that both SST and its receptor, SSTR2, are expressed locally within chicken thymus. The in vitro study showed that SST significantly inhibits IL-2 and concanavalin A (ConA) induced proliferation of thymocytes. In comparison with controls (medium containing IL-2 and ConA but without SST), addition of SST at 10-9 M and 10-6 M resulted in a nearly 20% decrease in proliferation (P < 0.01). The effects of somatostatin (SST) on the immune system, the role of SST on the gene expression of cytokines (IL-1, TGF, INF), chemokine receptors (CXCR4) as well as MHC-I components was assessed by real-time PCR. The question as to exactly which stimuli trigger the release of mediators such as somatostatin remains for future study. In addition, a complete inventory of all substances stored in the thymic LDCV and their effects on the developing T-cells when released in the microenvironment of the thymus are also questions that warrant further investigation.

Neuroendocrine cell populations were systematically studied and characterized in the thymus, an avian primary immune organ. The expression of the specific mRNAs for both Chromogranin A (CgA) and Carboxypeptidase E (CpE) in the thymus was first verified by RT-PCR. Additional evidence using immunofluorescent dual labeling, has demonstrated for the first time the co-existence of CgA and CpE in identical neuroendocrine cells at the protein level in a vetebrate primary lymphoid organ. These CpE- and CgA-positive cells were primarily found in the transition zone between the cortex and the medulla of the thymic lobules, an area known to contain numerous arterioles and to be heavily innervated by the autonomic nervous system, suggesting that these cell population can potentially receive input from each other, from the autonomous nervous system, from the circulation, or all of the above. (Neuro)endocrine messenger molecules produced by the thymic microenvironment, such as somatostatin (SST), seem to play a potentially important immunomodulatory role with regard to cell proliferation, differentiation, and migration, as well as cytokine production. The results showed that both SST and its receptor, SSTR2, are expressed locally within chicken thymus. The in vitro study showed that SST significantly inhibits IL-2 and concanavalin A (ConA) induced proliferation of thymocytes. In comparison with controls (medium containing IL-2 and ConA but without SST), addition of SST at 10-9 M and 10-6 M resulted in a nearly 20% decrease in proliferation (P < 0.01). The effects of somatostatin (SST) on the immune system, the role of SST on the gene expression of cytokines (IL-1, TGF, INF), chemokine receptors (CXCR4) as well as MHC-I components was assessed by real-time PCR. The question as to exactly which stimuli trigger the release of mediators such as somatostatin remains for future study. In addition, a complete inventory of all substances stored in the thymic LDCV and their effects on the developing T-cells when released in the microenvironment of the thymus are also questions that warrant further investigation.

Mémoire de Sage-femme : Médecine : Nantes : 2007. / Bibliogr.

No description available.

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 119-132).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 97-118).

Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas. / Made available in DSpace on 2018-08-17T07:03:20Z (GMT). No. of bitstreams: 1 Mansur_Eli_D.pdf: 1693041 bytes, checksum: a5b3d9f350100a50b16d835341ff4bc9 (MD5) Previous issue date: 2007 / Resumo: A leptina, hormônio com semelhança funcional e estrutural às citocinas, é conhecida por exercer, além das ações clássicas de controle da ingestão alimentar e termogênese, importantes funções na modulação das respostas do sistema imune. Alguns destes efeitos são dependentes da propriedade da leptina em modular a apoptose das células tímicas. Neste trabalho, utilizamos ratos Wistar para investigar os mecanismos moleculares envolvidos no controle, dependente da leptina, da apoptose no timo. A apoptose foi avaliada por citometria de fluxo e ELISA para determinação de nucleossomos, enquanto que a transdução do sinal foi avaliada por imunoprecipitação, imunoblot e microscopia confocal. O ObR estava expresso na maioria das células tímicas e a sua quantidade relativa reduziu-se progressivamente durante a maturação dos timócitos. A expressão do ObR estava co-localizada com JAK-2 e STAT-3, e a injeção aguda, in vivo , de leptina promoveu a fosforilação em tirosina de JAK-2 e o engajamento de STAT-3. O tratamento com leptina, também, levou à fosforilação em tirosina de IRS1 e fosforilação em serina de Akt. O tratamento crônico com leptina reduziu a apoptose tímica, e este efeito não foi inibido pelo AG490, um inibidor de JAK, mas foi significativamente inibido por LY294002, um inibidor de PI3-Quinase, e por um oligonucleotídeo antisense para IRS1. Portanto, a leptina inibe a apoptose em células tímicas via um mecanismo independente da ativação de JAK-2 mas dependente do engajamento da via IRS1/PI3-Quinase / Abstract: The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we employed Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, while signal transduction was evaluated by immunoprecipitation, immunoblot and confocal microscopy. The ObR was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was co-localized with JAK-2 and STAT-3, and an acute, in vivo , injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of STAT-3. The treatment with leptin also led to the tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG490 but was significantly inhibited by the PI3-kinase inhibitor LY294002 and by an antisense oligonucleotide to IRS1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS1/PI3-kinase pathway / Doutorado / Medicina Experimental

Thymic negative selection has been identified as a crucial checkpoint in thymocyte development that purges the T-cell repertoire of autoreactive T cells through apoptosis of the cells after strong T cell receptor (TCR) stimulation. It has been well established that efficient thymic negative selection is required to prevent severe monogenic autoimmune diseases, such as Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy. The involvement of negative selection in other T cell-mediated autoimmune diseases remains unclear. This is largely due to the lack of fully-humanized physiological models for the study of human thymic negative selection. In the work presented here, I aim to study human thymic negative selection in healthy control (HC) immune systems and to determine whether negative selection is impaired in immune systems from individuals with Type 1 diabetes (T1D), a T cell-mediated autoimmune disease. To facilitate these studies, I developed novel humanized mouse and organ culture models. These models built on the previously described Personalized Immune (PI) mouse model [1]. The PI mouse model allows for the rederivation of a fully humanized immune system from a human donor by transplanting hematopoietic stem cells (HSC) and progenitors from an individual adult donor and a human fetal thymus fragment to immunodeficient mice. The HSCs then reconstitute all immune cell lineages, including T cells which develop in the human thymus fragment. This model is extremely powerful because it allows for the study of a human’s immune system in a model that is conducive to experimental replicates and interventions, unlike studies done directly on human patients. To optimize this model for the study of thymic negative selection, I developed two other PI models. The first is the TCR-transgenic PI thymic organ culture (TOC) model. This model allows for the study of the selection of a specific TCR in a culture system combining human HSCs and thymus fragments. The second model is the TCR-transgenic PI mouse model. This model allows for the study of the thymic selection of a specific TCR in a fully humanized in vivo model. The work presented here utilized these three powerful PI models to interrogate the thymic negative selection process in human health and disease at a depth not previously possible. Using these models, we demonstrated the first evidence for thymic negative selection of an insulin-reactive TCR that recognizes a naturally expressed antigen in healthy human immune systems. These studies also demonstrated that robust negative selection requires HSCs expressing the HLA-restriction element of the TCR, and without the expression of that HLA on HSCs, negative selection is reduced and performed in later stages of thymic development. When comparing the phenotypic and functional characteristics of thymocytes undergoing negative selection in HC and T1D immune systems, T1D thymocytes in some immune systems had differential expression of TCR-signaling and negative selection markers and resistance to apoptosis and cell death after strong TCR stimulation. Studies on the negative selection of a specific insulin-reactive TCR in healthy and T1D immune systems demonstrated that in healthy immune systems central tolerance to this TCR involved a combination of negative selection and T regulatory cell conversion. This is the first demonstration of combined tolerogenic induction in the human immune system. In contrast, some T1D immune systems demonstrated impaired negative selection of this insulin-reactive TCR and impaired conversion of these autoreactive T cells to T regulatory cells. Further, when comparing the gene expression profile of HC and T1D thymocytes undergoing negative selection, there are multiple genes important in thymic selection and apoptosis that are differentially regulated. Overall, this data provides unique insights into the process of thymic negative selection in healthy immune systems. It also provides the first evidence that thymic negative selection is impaired in some T1D immune systems and this impairment is possibly driven by differential gene expression. The models developed will allow for further study into human thymic selection in health and disease. These findings answer the important question of whether thymic negative selection is impaired in autoimmune disease, which has been debated in the field of T1D research and the wider immunology field. More importantly, it opens the door to targeting of the thymic negative selection pathway with therapeutics for T1D and other autoimmune diseases.