Browsing by Subject "Thymus"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Developing novel strategies to enhance thymic recovery and T-cell reconstitution following bone marrow transplantation.
    (2009-05) Kelly, Ryan Michael
    Allogeneic HSCT is a valuable treatment option for many malignant and nonmalignant disorders. A significantly limiting factor for a favorable outcome following HSCT is the prolonged T-cell deficiency following transplant, which is primarily due to thymic injury caused by the intense chemotherapy/radiation-conditioning regimen given prior to transplant. The submitted work details the development of novel approaches to restore thymic function and enhance T-cell reconstitution following bone marrow transplantation (BMT). The preclinical research described in this dissertation investigates the therapeutic potential of combinatorial administration of keratinocyte growth factor, androgen regulators and general radioprotectants in restoring thymic function and T-cell reconstitution following BMT. The data suggest that pre-conditioning treatment of BMT recipients with combinations of these agents lead to rapid and durable restoration of thymic function and accelerated peripheral reconstitution of donor-derived, naïve CD4 and CD8 T-cells. Importantly, enhanced T-cell reconstitution correlates with superior antigen-specific CD4 and CD8 T-cell responses in vivo. This work also describes research aimed at characterizing the kinetics of depletion and recovery of thymic epithelial cells (TEC) following BMT and elucidating the role of thymocyte:TEC crosstalk in promoting TEC regeneration. A more thorough understanding of this process will allow for the identification of more focused targets for therapies aimed at promoting thymic and T-cell reconstitution following BMT. Taken together, this work has generated novel findings that will advance the field of immune reconstitution following bone marrow transplantation.
  • Thumbnail Image
    Item
    Development Of Regulatory T Cells Capable Of Maintaining Immune Homeostasis
    (2020-09) Owen, David
    The adaptive immune response, comprised of both T cells and B cells, is essential to control infections and eliminate transformed cancer cells. The success of the adaptive immune system relies on the ability to discriminate self from non-self-antigens. The thymus is the site of selection for T cells, where self-reactive T cells are eliminated, generating a non-self focused T cell compartment. However, this selection process is leaky and potentially pathogenic cells do escape thymic, or central, tolerance. Thus, a population of suppressor cells termed regulatory T cells (Treg cells) co-evolved in order to keep these self-reactive escapees in check. Treg cells that develop in the thymus as part of central tolerance induction are a critical population of T cells that are required to maintain immune homeostasis and prevent autoimmunity. Without intervention, mice or humans that lack the ability to generate Treg cells die shortly after birth from widespread autoimmune-mediated tissue destruction. Further, neonatal thymectomy in mice causes the development of an autoimmune wasting phenotype. These observations highlight the importance of thymic Treg cell selection in immune homeostasis. Thymic Treg cell development occurs via a two-step process. Step one involves developing CD4+ thymocytes receiving strong T cell receptor (TCR) stimulation via engagement of thymic self-antigens, leading to upregulation of CD25, the high affinity subunit of the IL-2 receptor, or FOXP3, the lineage defining transcription factor of Treg cells, generating either CD25+ or FOXP3lo Treg cell progenitors (TregP). Step two is driven by encounters between TregP cell and intrathymic STAT5 activating cytokines, predominantly IL-2, leading to co-expression of CD25 and FOXP3. These CD25+FOXP3+ cells represent fully mature Treg cells that disseminate from the thymus to mediate immune tolerance. While the framework of this two-step development process is understood, many details of each step remain incompletely understood. This thesis addresses several aspects of thymic Treg cell development. First, we identify that T cells are the critical source of IL-2 required to drive Treg differentiation. Second, we provide evidence that CD25+ and FOXP3lo TregP arise via distinct selection programs and contribute functionally distinct TCRs to the mature Treg compartment. Third, using single-cell RNA-sequencing analysis of conventional and Treg lineage thymocytes we provide a more detailed analysis of transcriptional signatures and intermediates of thymic Treg development. Finally, we gathered preliminary data to better understand the heterogeneity and function of recirculating or resident thymic Treg cells. Developing a holistic understanding of Treg development is essential to discern the etiology of immune disorders and properly modulate Treg cells to treat autoimmune disease, infections and cancer.
  • Thumbnail Image
    Item
    Evaluating thymocyte negative selection within the polyclonal population
    (2019-09) Breed, Elise
    The development of a self-tolerant and effective T cell receptor repertoire is dependent on interactions coordinated by various antigen presenting cells (APC) within the thymus. T cell receptor–self-peptide–MHC interactions are essential for determining T cell fate, where high affinity interactions can result in clonal deletion or regulatory T (Treg) cell differentiation of potentially autoreactive T cells. The APCs that provide these signals have distinct localization, different antigen processing features, and can provide different co-stimulatory signals that are also critical to these selection processes and may distinguish the ultimate fate of a T cell. Clonal deletion and Treg differentation of T cells specific for self-antigens in the thymus have been widely studied, primarily by approaches that focus on a single receptor (using TCR transgenes) or a single specificity (using pMHC tetramers). However, little is known about how distinct APCs coordinate clonal deletion and Treg cell development at the population level. Here, we report an assay that measures cleaved caspase 3 to define clonal deletion at the population level. This assay distinguishes clonal deletion from apoptotic events caused by neglect and approximates the anatomic site of deletion using CCR7. This approach showed that 78% of clonal deletion events occur in the cortex in mice. Medullary deletion events were detected at both the semi-mature and mature developmental stages, although mature events were associated with failed Treg cell induction. Using this assay, we showed that bone marrow derived APC drive approximately half of deletion events at both stages. We also found that both cortical and medullary deletion rely heavily on CD28 co-stimulation. We further assessed the contribution of distinct APC subsets to clonal deletion and Treg cell selection using cell type ablation or deficiency. We found that total deletion and nascent Treg cell events were not altered in the absence of B cells, pDC, or XCR1+ cDC1. In an effort to eliminate SIRPa+ cDC2, we discovered that a fraction of thymic SIRPa+ cDC2 express the lectin CD301b. These cells resemble the type 2 immune response-promoting CD301b+ DC that are present in skin draining LN. CD301b expression was localized primarily within the thymus medulla and depended on IL-4R. Deficiency of these IL-4 and IL-13 signaled cDC2 caused a measurable reduction in clonal deletion events, suggesting a non-redundant role for tolerance induction. These findings demonstrate useful strategies for studying clonal deletion and nascent Treg cell development within the polyclonal population. Additionally, they provide valuable insight into how and when thymocytes undergo clonal deletion as they traverse through the thymus and interact with distinct APC during development.
  • Thumbnail Image
    Item
    How lipid specific T cells become effectors
    (2019-04) Wang, Haiguang
    Invariant natural killer T (iNKT) cells are composed of at least three functionally distinct subsets, NKT1, NKT2 and NKT17. Through selective activation of these three iNKT effector subsets, iNKT cells can modulate immune responses and tissue homeostasis in different fashions. However, the developmental steps that drive iNKT cells into functional distinct subsets have not been elucidated, and thus their potential to be utilized in anti-cancer or autoimmune immunotherapies has not been realized, despite the fact that iNKT stimulatory lipids are well-tolerated in human trials. My dissertation research aims to fill this knowledge gap by investigating the following aspects of iNKT biology: 1) characterizing the multipotent progenitor for the iNKT effector subsets (in chapter 2); 2) isolating the critical factors that determine how individual iNKT subsets are derived, with a focus on NKT2 cells (in chapter 3); 3) characterizing how distinct iNKT effector subsets specifically modulate protective host immune responses (in chapter 2 & 3); and 4) technical improvement in advancing more accurate analysis of ex vivo iNKT cells (in chapter 4). Firstly, in chapter 2, I demonstrate that the small proportion of thymic iNKT cells that express CCR7 represent a multi-potent progenitor pool that gives rise to effector subsets within the thymus. These CCR7+ iNKT cells also emigrate from the thymus in a Klf2 dependent manner, undergo further maturation after reaching the periphery. Furthermore, Ccr7 deficiency impaired differentiation of iNKT effector subsets and localization to the medulla. Parabiosis and intra-thymic transfer showed that thymic NKT1 and NKT17 were resident-they were not derived from and did not contribute to the peripheral pool. Finally, each thymic iNKT effector subset produces distinct factors that influence T cell development. Secondly, previous studies showed IL-4 is produced by NKT2 cells in the thymus, where it conditions CD8+ T cells to become “memory like” amongst other effects in the steady state. However, the signals that cause NKT2 cells to constitutively produce IL-4 remain poorly defined, where in the chapter 3, these signals were investigated. Using histocytometry, IL-4 producing NKT2 cells were localized to the thymic medulla, suggesting medullary signals might instruct NKT2 cells to produce IL-4. Moreover, NKT2 cells receive and require TCR stimulation for continuous IL-4 production at steady state, since NKT2 cells lost IL-4 production when intra-thymically transferred into Cd1d deficient recipients. In bone marrow chimeric recipients, only hematopoietic, but not stromal APC, provided such stimulation. Furthermore, using different Cre-recombinase transgenic mouse strains to specifically target CD1d deficiency to various APC, together with the use of diphtheria toxin receptor (DTR) transgenic mouse strains to deplete various APC, we found that macrophages were the predominant cell to stimulate NKT2 IL-4 production. Lastly, it has been recently shown that high extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT. The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets is unclear, however, as is the impact of recovery from other nonlymphoid sites. Therefore, in the chapter 4, I showed NKT1 cells express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17 cells. Furthermore, I demonstrated that ARTC2.2 blockade enhanced NKT1 recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of iNKT cells. Therefore, short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry–based phenotyping and enumeration of murine iNKT from nonlymphoid tissues, and it represents a crucial step for functional studies of this population. Altogether, I believe the findings here provide a clearer understanding of how the lipid specific iNKT cells become effector subsets as well as a technical improvement for accurate analysis of these cells.
  • Thumbnail Image
    Item
    Mechanisms of Thymic Involution and Therapies to Prevent or Treat the loss of Thymic Epithelial Cells
    (2016-09) Smith, Michelle
    The thymus has great importance to human health as naïve T cells cannot be generated in its absence. The composition and organization of its specialized microenvironment are the foundation of the function of the thymus. In particular, thymic epithelial cells and the segregation of their subtypes, cortical thymic epithelial cells and medullary thymic epithelial cells, into distinct areas are required for positive and negative selection of developing thymocytes. However, these cells can be lost over time through natural age-related processes and through acute injury such as chemotherapy or radiation. The purpose of this thesis is to increase the understanding of how these losses can occur and to investigate therapies which may prevent or treat these losses. We first focus on a stem cell-based therapy for the improvement of thymopoiesis following radiation-based injury to the thymus. In it are discussed findings which describe different outcomes which may be achieved by selection of specific input populations. The disruption which the radiation-induced damage has on intrathymic migration of progenitors is also highlighted as well as the temporary nature of their stimulatory effects on thymic epithelial cells. Next, we also focus on thymic epithelial cell loss, but precipitated by advancing age. To facilitate these studies, a model of accelerated aging caused by deficiency in the gene klotho was used. We found that klotho deficiency did not impart an intrinsic defect in TEC longevity but that elements of the systemic aging environment were responsible for accelerated TEC loss in a TEC non-autonomous fashion. Specifically, high levels of vitamin D which accumulate in these mice were implicated in the induction of abnormal TEC apoptosis. Together the findings presented here advance the understanding of mechanisms which may be responsible for the loss of thymic epithelial cells, either age-related or clinically induced. Two strategies for the prevention or treatment of TEC loss are also discussed.
  • Thumbnail Image
    Item
    Thymic interferons and protein O-GlcNAcylation in regulatory T cells: two tales of T cell tolerance
    (2021-03) Salgado Barrero, Oscar
    Immune tolerance mechanisms prevent the development of immune responses directed to the host. This is especially important for the adaptive immune system, whose potent and long-lasting responses would be extremely deleterious to the host if misguided. This work explores two aspects of immune tolerance: the role of protein O-GlcNAcylation in regulatory T (Treg) cells and the importance of interferons during T cell tolerance development in the thymus. In chapter 2 of this document, we show that the posttranslational modification by O- linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. These data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells. In chapter 3, we characterized the expression of interferons in the thymus. We found that developing thymocytes displayed a type I IFN signature that was mainly dependent on IFN-β. Using Ifnb tdtomato and luciferase reporter mouse strains, we found expression in a small population of medullary thymic epithelial cells (mTEC), which was AIRE dependent and peaked at 2-3 weeks of age. To study the cellular response to thymic interferon, we used an Mx1gfp reporter mouse strain and report that numerous thymic cell populations respond constitutively to IFN in vivo. The response in some cell populations was not abrogated unless both IFNAR and IFNLR, or STAT1 were deficient, suggesting that both type I and type III IFNs are at play. Indeed, single cell RNA sequencing analysis revealed dramatic transcriptional changes in all thymic APCs in IFNAR/ IFNLR deficient mice. These results show that steady state type I and type III IFN signaling drives a gene-expression program in thymic APCs that shapes the thymic microenvironment.