Browsing by Subject "IL-4"

Now showing 1 - 4 of 4
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    Derivation, maintenance, and functions of virtual memory cells.
    (2011-09) Akue, Adovi Dodji
    Memory phenotype CD8+ T cells are typically thought to have undergone an immune response to foreign antigen and to have differentiated from antigen-specific precursors in the naïve pool. However, using a peptide-MHC I tetramer enrichment technique, we identified foreign antigen-specific memory-phenotype CD8+ T cells in unimmunized mice. These cells (termed "virtual memory" T or VM cells) were observed in mice maintained in both specific-pathogen- and germ-free (SPF and GF respectively) housing. This thesis focuses on the relationship between VM cells and "conventional" memory cells: memory cells arising from homeostatic proliferation (HP), and innate-like memory CD8+ T cells such as IL-4 bystander memory CD8+ T cells. Our data indicate physiological HP and IL-4-driven bystander processes are the main mechanisms that drive the generation of VM cells and not the exposure to foreign antigens. VM cells arise in the periphery during the neonatal period and are maintained long term. We also show that VM cells respond in vitro to innate cytokines (similar to conventional memory CD8+ T cells) and they outcompete antigen-specific naive CD8+ T cells in in vivo responses. Overall our observations suggest that VM cells arise out of normal homeostatic and IL-4-driven bystander processes in unimmunized SPF and GF mice, and express at least some memory-like capabilities.
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    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.
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    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.
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    Identifying Mechanisms And Biomarkers Predictive Of Efficacy Of Vaccines Against Opioid Use Disorders And Overdose
    (2022-08) Crouse, Bethany
    Opioid use disorders (OUD) and overdose are public health crises that are worsening despite the availability of approved pharmacotherapies. Active immunization with anti-opioid conjugate vaccines is a novel therapeutic strategy to treat OUD and prevent overdose. To date, clinical studies suggest that efficacy of anti-drug conjugate vaccines is limited to a subset of individuals who can produce optimal antibody responses. To increase positive treatment outcomes and clinical success, this research program investigated several complementary strategies to increase OUD vaccine efficacy. First, mechanisms of optimal anti-opioid vaccine response are investigated by elucidating the immunological mechanisms behind a previously established interleukin-4 (IL-4) mediated increase in vaccine efficacy. These studies found that depletion of IL-4 resulted in a Type I IL-4R mediated increase in germinal center formation and germinal center T cell response which leads to increased opioid-specific antibody secreting cells, and that vaccine efficacy is dependent on a balanced Th1/Th2 T cell response in mice. Next, these results provided a blueprint for next generation anti-fentanyl vaccine formulations incorporating novel adjuvants targeting toll-like receptors (TLRs). These data show that a TLR7/8 agonist adjuvant increases vaccine efficacy in rodent and porcine models of fentanyl misuse and overdose. Third, vaccine design and immunization paradigms were assessed to optimize the efficacy of a novel carfentanil vaccine alone and in combination with a lead fentanyl vaccine. Longer linker lengths and a co-administered bivalent immunization strategy were associated with increased vaccine efficacy. Then, environmental factors contributing to the immune response are investigated by testing whether changes in the gastrointestinal microbiome would affect vaccine efficacy and whether these specific changes in the microbiome could be utilized as biomarkers. These studies revealed that changes in the microbiome in specific pathogen free or immune-experienced rodents did not affect efficacy of anti-oxycodone or anti-fentanyl vaccines. Finally, exploratory studies were performed to identify putative biomarkers that may be predictive of anti-opioid vaccine response in preclinical and clinical investigations. These studies indicate that pre-immunization concentration of IL-4 is correlated with vaccine efficacy in genetically diverse mice, and that specific cytokines may be of interest as indicators of immune response in human patients. Overall, the findings outlined in this research program support the use of novel adjuvants and predictive biomarkers to increase clinical efficacy of vaccines to treat OUD and overdose.