Browsing by Subject "Tolerance"
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Item Development Of Regulatory T Cells Capable Of Maintaining Immune Homeostasis(2020-09) Owen, DavidThe 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.Item The effects of polydextrose and soluble corn fiber on laxation and satiety in healthy human subjects(2012-09) Timm, Derek AllenDietary fiber from whole foods has long been known to regulate bowel function; however it is essential to confirm this in isolated or synthesized fibers. Stool weight, gastrointestinal transit time, and stool consistency are all ways to measure changes in bowel function in humans and can be used to evaluate the efficacy of dietary fibers. Gastrointestinal tolerance is also of great concern since consumer acceptability of dietary fiber is related to intolerance issues. Furthermore, since observational and epidemiological data suggests dietary fiber reduces energy intake, which may be modulated by changes in postprandial satiety, we also investigated acute satiety using visual analog scale (VAS) and food intake via food diaries. Therefore, we investigated the influence of 20 grams per day for 10 days of the functional fibers Polydextrose (PDX) and Soluble Corn Fiber (SCF) compared a low fiber control on bowel function. Thirty-six healthy men and women completed this randomized, double-blind, placebo-controlled study. A two-week washout period was completed between each treatment period. Results show both PDX and SCF significantly increased stool weight compared to the control treatment. In contrast, whole gut transit time was not difference among the treatments. Stool pH was significantly lower for PDX compared to the control treatment. PDX caused a significantly looser stool than SCF and control. Flatulence and stomach noises were significantly increased by the fiber treatments compared to the control. Satiety was not difference among the treatments as measured by a VAS. No differences were observed in energy intake among the treatments.Item Evaluating thymocyte negative selection within the polyclonal population(2019-09) Breed, EliseThe 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.Item The function and utility of self-specific CD8 T cells(2018-02) Nelson, ChristineSelf-specific CD8 T cells have the potential to provide great benefit, but also to cause great harm. This thesis research seeks to uncover the mechanisms controlling CD8 T cell tolerance to self and tumor antigens. We show that the induction of CD8 T cell tolerance is dependent on inhibitory receptor signaling, but the maintenance of tolerance is not. Thus, only newly primed self-specific CD8 T cell are amenable to immune checkpoint blockade activation. We establish CD8 T cell tolerance as an active state of differentiation that is dependent on suppressed antigen-sensing. We demonstrate that robust stimulation with self-antigen and inflammation induces avidity maturation of the self-specific CD8 T cell repertoire, which serves to renew susceptibility to immune checkpoint and aid in anti-tumor responses. We validate that endogenous self-specific CD8 T cells exist within the immune repertoire and can expanded by similar vaccination methods. Expanded self-specific CD8 T cells generate tissue resident memory in non-lymphoid tissues and secondary lymphoid organs, that are phenotypically distinct from non-self-specific populations. Finally, we show that self-specific CD8 T cells synergize with tumor neo-antigen specific CD8 T cells in the treatment and prevention of cancer. This data provides significant insight into the requirements for the induction of self and anti-tumor T cell responses.Item Insights on CD8+ T cell self-tolerance from studies of a polyclonal melanocyte-specific population using a novel mouse model(2021-06) Truckenbrod, EmilySelf-specific CD8+ T cells can escape clonal deletion, but the properties and capabilities of such cells and the mechanisms restraining them require further clarification. Much of the data on self-tolerance originate from mice with genetically-manipulated TCR repertoires, and there is a need for improved models that better recapitulate normal physiology. We established a novel self-tolerance model focused on polyclonal CD8+ T cells specific for tyrosinase-related protein 2 (Trp2), a melanocyte enzyme encoded by the dopachrome tautomerase gene (Dct). We examined mice expressing (wild-type [WT]) or lacking (Dct-/-) this enzyme; Trp2 represents a self-antigen in WT mice and a foreign antigen in Dct-/- mice. Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the population size was only slightly reduced in WT relative to Dct-/- mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+ proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Examination of the T cell receptor (TCR) repertoire as characterized by single-cell sequencing revealed clonotypes shared both within and between the WT and Dct-/- Trp2/Kb-specific populations early after activation. Clonal expansions were more frequent among the Dct-/- repertoire, suggesting that higher-performing cells were more likely to be eliminated in WT animals. Our physiologically-relevant model demonstrates that CD8+ T cells can exhibit functional, cell-intrinsic self-tolerance while sharing many features with non-tolerant cells. This model will be useful in further mechanistic investigation of self-tolerance that may ultimately lead to more effective therapeutic manipulation of the immune system.Item Myeloid derived suppressor cells in dogs with cancer: phenotype, function and clinical implications(2014-06) Goulart, Michelle RodriguesMyeloid-derived suppressor cells comprise phenotypically heterogeneous population of myeloid cells at different stages of differentiation endowed with potent immunosuppressive activity. Abnormal accumulation of MDSC in tumor models and cancer patients produce profound immune suppression, severely impairing T cell antitumor immunity, contributing to angiogenesis, cell invasion and metastasis, and constitute a major hurdle in achieving successful immunebased therapies. Understanding the mechanism that drives MDSC expansion and enhances function in humans and dogs is crucial for the development of efficacious immunotherapy. Studies in dogs with several tumor types, including sarcoma, carcinomas, mast cell tumors and gliomas confirmed MDSC expansion in the peripheral blood of dog cancer patients. MDSC have been identified in dogs using the combination of three-marker phenotype CD11b+CD14-MHCII-cells for granulocytic and CD11b+CD14+MHCII-cells for monocytic subsets. Granulocytic MDSC accumulated in the peripheral blood of dogs with advanced sarcoma, carcinomas and mast cell tumors, co-purified with peripheral blood mononuclear cell (PBMC) fraction and expressed polymorphic mononuclear morphology. This subset of cells showed the ability to efficiently inhibit T cell proliferation and IFN-γ secretion of autologous T cells, as well as allogenic T cells from healthy dogs, and expressed ARG1, iNOS2, TGF-β and IL-10. Monocytic MDSC also demonstrated potent ability to suppress T cell proliferation and preferentially accumulated in the peripheral blood of dogs with glioma. Elevated levels of arginase activity found in the serum of dogs with glioma could potentially be due to the presence of elevated numbers of MDSC. Evaluation of the anti-mouse Gr1 antibody for MDSC staining and identification revealed that does not cross react and therefore is not suitable for canine cells.Item Peanut allergic children residing in Olmsted County, MN: an examination of the prevalence in 2007, incidence rates from 1999-2007 and association between peanut-specific IgE level, tolerance and reaction severity.(2011-05) Rinaldi, Maria CarolINTRODUCTION Peanut allergy is a significant concern due to increased prevalence over the last few decades, potential severity of a reaction, and the large percentage of children who do not acquire tolerance. This dissertation evaluated rates of diagnosis over time and examined whether peanut-specific IgE level has clinical relevance regarding tolerance and reaction severity. METHODS Data on all children with a peanut allergy diagnosis between 1999-2007 in Olmsted County, MN was collected using the Rochester Epidemiology Project. The first study estimated the prevalence in 2007 and incidence rate of peanut allergy diagnoses from 1999-2007. This study further examined whether the number of peanut allergy diagnoses from 1999-2007 varied by reaction severity. The second study estimated the percentage of children that developed tolerance to peanuts and assessed the association between peanut-specific IgE level and tolerance. The third study evaluated the association between peanut-specific IgE level and reaction severity. RESULTS The 2007 prevalence of peanut allergy was 0.59%. There were statistically significant lower rates of peanut allergy diagnoses among females (82.0%) as compared to males and among children aged 3-17 years (99.9%) as compared to those aged 0-2 years. There was a significant 1.7-fold increase in peanut allergy diagnoses from 3.84 cases per 10,000 children in 1999-2001 to 6.53 per 10,000 children in 2005-2007. There was not a significant difference in the number of children having had mild as compared to moderate/severe reactions over time. In this sample, 16.4% developed tolerance to peanuts. Children with peanut-specific IgE class levels 4-6 as compared to those with levels 1-3 had a significant 91.0% reduced likelihood to develop tolerance after adjustment for number of atopic conditions and a 2.15 non-significant greater odds for a moderate/severe reaction after adjustment for age at diagnosis. CONCLUSION The incidence rate of peanut allergy increased irrespective of severity of first reaction and most children did not acquire tolerance. The majority of those diagnosed with peanut allergy were males and 0-2 years old. Peanut-specific IgE level was not associated with initial reaction severity, but was found to be a useful prognostic tool for tolerance.Item Thymic interferons and protein O-GlcNAcylation in regulatory T cells: two tales of T cell tolerance(2021-03) Salgado Barrero, OscarImmune 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.Item Tolerance, governance, and surveillance in the Jim Crow South: Asheville, North Carolina, 1876-1946(2013-02) Epstein, Seth Edward DavidThis dissertation argues that logics of tolerance were central to emerging forms of urban governance in the New South tourist locale of Asheville, North Carolina between the late 19th and mid-20th centuries. White authorities' practices of "race relations," the development of civic sites of historical memory, and the regulation of disorderly spaces worked to distribute the responsibility of surveillance to many actors. Most significantly, objects of suspicion were enlisted and enlisted themselves in networks of authority as a means to police and, hopefully, transcend the danger to urban order they themselves embodied. These networks were hierarchical. Their priorities and the relations between actors within them were shaped and supported by white authorities' political privilege to formulate racialized, gendered, and class-conscious definitions of deviance. They were also distributive, as their operation depended on the efforts of multiple participants. The forms of governance organized around techniques of tolerance did not dispel white authorities' suspicion, nor aim to. Instead, the projects considered here created opportunities to make that suspicion operable and regularize its management. By focusing on one city, this dissertation is able to demonstrate how the development, maintenance, and changes in networks of tolerance played a key role in making and remaking both place and space in Asheville. Scrutinizing these networks is essential for understanding how tolerance both created space for civic participation and sharply curtailed what would be tolerated within it. Through variously articulating, critiquing, and performing the expectation of surveillance, African Americans, Jews, and white Christians sought to redefine the boundaries of tolerable difference in urban spaces as well as the meanings of blackness, Jewishness, and whiteness. This dissertation employs insights drawn from cultural geography and government studies to interrogate tolerance as a technique of management. It therefore newly historicizes the emergence of tolerance as a national civic value in the interwar period and reassesses its analytical value to urban history.Item Visualizing CD8+ T cell responses to foreign and self-antigen(2017-08) Thompson, EmilyCD8+ T cells can recognize any infected cell of the body, making them essential in the immune response against intracellular pathogens. A critical function of CD8+ T cells is the directed killing of target cells through cytolysis. This mechanism is dependent on direct cell-cell contact, which makes the migratory capacity of CD8+ T cells paramount to their successful immune response. The small intestine (SI) is the biggest mucosal surface between the host and the environment. The immune system in this compartment must actively eliminate infection while maintaining tolerance to normal flora, self, and food-antigen. Using two-photon laser scanning microscopy, I evaluated foreign- and self-specific CD8+ T cell motility in the SI. I found that foreign-antigen specific CD8+ T cell behavior varied throughout infection, and was independent of the αE integrin. Interestingly, self-specific CD8+ T cells were initially reactive to self-antigen in vivo but this behavior was altered after further tolerance induction. These studies inform our understanding of the requirements for effective CD8+ T cell immunosurveillance in the SI. I also evaluated what characterizes and contributes to a self-specific CD8+ T cell response to protein in the SI. Using a heterologous prime-boost-boost (HPBB) approach, I generated functional self-specific CD8+ T cells. This response matured throughout boosting, showing the potential of self-specific CD8+ T cells. I also used HPBB to evaluate foreign-antigen specific CD8+ T memory development and showed that the time interval between each boost impacts CD8+ T cell memory longevity.