ABSTRACT Background Interleukin-7 (IL-7) is a cytokine crucial for T-cell development and homeostasis. IL-7 is thought to be a limited resource, and its interaction with the IL-7 receptor (IL-7R) has effects on increasing cell survival, proliferation and cytolytic function. Considering the roles of IL-7, it is no surprise that the expression of the IL-7 receptor alpha chain (CD127) is tightly regulated. Despite increased levels of soluble CD127 (sCD127) being detected in a number of disease states and being associated with disease activity, the biological function of sCD127 and its clinical relevance remains to be established. In this study, I explore the post-translational mechanisms leading to the release of the soluble form of CD127 receptor through IL-7 and αCD3/αCD28 stimulation. Here I specifically established two different mechanisms by which CD127 is processed; shedding of the receptor ectodomain and clipping. Results In CD8+ T-cells, IL-7 plus TcR stimulation resulted in an increased release of sCD127. Here I found that matrix metalloproteases (MMPs), in particular MMP-9, have a role in the proteolytic clipping of CD127 resulting in the release of sCD127. In addition, I found that IL-7 plus TcR stimulation resulted in an increase in MMP activity and this activity was particularly dampened when MMP-2 and -9 inhibitors were used. I also found that neither MMP-3 nor cysteine and serine proteases seem to be directly involved in the generation of sCD127. Using a biotinylation assay I found that CD127 is being shed from the surface of CD8+ T-cells as well as thymocytes through a MMP-independent mechanism. Conclusion These results demonstrate that MMPs (in particular MMP-9) have a role in the generation of sCD127. Further studies are required to determine the specific sheddase responsible for the ectodomain shedding of CD127, as well as the details behind the regulation of MMP-9 activity both in CD8+ T-cells and thymocytes. A thorough understanding of these mechanisms will aid in the development of alternative and more specific strategies to control IL-7 mediated processes in both normal and disease states.

Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system. The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation. Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis. This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.

The Interleukin (IL)-17 cytokine family, expressed by T helper (Th)17 cells and γδ T cells, plays pivotal roles in adaptive immune responses. They have been implicated in autoimmune and allergic diseases as well having roles in bacterial and fungal clearance. Importantly, IL-17 producing γδ T cells have been shown to be critical for the development of adaptive Th17 responses in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis. Interestingly, natural ligands of the aryl hydrocarbon receptor (AhR) are known to influence the development of Th17 cells. It has been shown previously that prolonged topical exposure of mice to the contact allergen 2-4 dinitrochlorobenzene (DNCB) or to the respiratory sensitiser trimellitic anhydride (TMA) causes the preferential development of a preferential T helper (Th)1 or Th2 response, respectively. The presence of the novel IL-17 family cytokines and their cellular source was investigated following both single and prolonged exposure to allergen. Exposure only to the contact allergen DNCB resulted in up-regulation of the expression of IL-17 by dermal γδ T cells. It was shown also that topical application of a range of contact allergens and of the respiratory allergen TMA resulted in IL-17 expression by γδ T cells in the lymph node draining the site of exposure. However, differential kinetics were observed between the two classes of allergens. Exposure to the contact allergens resulted in rapid expression of IL-17 within 6-16 h, whereas the respiratory allergen displayed considerably delayed kinetics, with maximal levels detected 48 h post exposure. Treatment with DNCB only was shown to be associated with the development of Th17 cells following prolonged exposure to chemical allergen. Thus DNCB provoked a Tc1/Th1/Th17 profile, in contrast prolonged exposure to TMA resulted in a very selective Th2 cytokine pattern. The influence of γδ T cells on polarised responses to chemical allergens was investigated also using γδ T cell knockout mice; here the adaptive Th17 response induced by DNCB was completely abrogated. These data demonstrate that the absence of IL-17 production by γδ T cells during the early innate immune response affects the subsequent adaptive Th17 response stimulated by chemical contact allergens. Finally, the importance of the affinity of the AhR for endogenous ligands during in vitro Th17 polarisation was assessed. Using three strains of mice with differential AhR affinities the contribution of ligation of these receptors in Th17 cell development was investigated. In all three strains AhR ligation was required for optimal polarisation of Th17 cells, even in strains that are reported to express a low affinity receptor. These data suggest that across a range of receptor affinities, including low affinity receptors analogous to that of humans, endogenous AhR ligands may play a major role in driving Th17 cell differentiation, regardless of receptor phenotype.

The maturation of cytotoxic T lymphocyte (CTL) effectors from CTL precursors (CTLp) requires specific signals mediated through cellular interactions and soluble factors. The most studied factor is T cell growth factor (TCGF) which is also termed interleukin-2 (IL-2). This lymphokine is produced by T helper cells (TH) and induces activated CTLp to proliferate and differentiate. However, in the absence of mitogen or antigen stimulation, IL-2 alone cannot induce CTL (except in the case of very high cell density). A factor is described that is found in the supernatant of 4-β-phorbol-12-myristate-13-acetate (PMA)-induced EL4 cells that can polyclonally activate CTL in the presence of IL-2. This factor elutes at 27 kilodaltons (KDa) on a G-100 column, and its target cell includes T cells of the Thyl⁺ Lyt2⁺ L3T4⁻ phenotype. The factor increases the frequency of IL-2 receptor expressing cells within a population, thereby increasing the response to IL-2. It is suggested that this factor acts through an alternative pathway of CTL activation which is independent of specific stimulation by antigen. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

When injected with live P815 tumour cells, DBA/2 mice developed cytotoxic cells reactive to the tumour. In addition, T helper cells from tumour-bearing mice enhanced the iri vitro generation of cytotoxic cells from normal DBA/2 spleen cells. The helper cells had the following properties (1) expression of the Thy-1.2 antigen; (2) resistance to y-radiation; (3) specific enhancement of the cytotoxic response to P815; (4) detectability in P815-bearing mice at the peak of cytotoxic cell activity; (5) activity in the early phase of cytotoxic cell activation. In parallel to the development of helper cell activity, suppressor cells were generated which suppressed the cytotoxic response to P815. These suppressor cells were removed by pre-treating mice with low doses of cyclophosphamide. High doses of cyclophosphamide reduced the cytotoxic response to both P815 and C57B1/6 alloantigens. Cyclophosphamide treatment reduced the frequency of cytotoxic precursor cells directed against P815, and an antigen-reactive helper cell involved in interleukin 2 production. Both interleukin 2 and thymocytes from P815-primed mice, restored the cytotoxic response against P815, to normal levels. Twenty six percent of animals primed with tumour cells cleared a challenge dose of P815 faster than unprimed control mice. Of these, 88% survived longer than the control animals. Eighteen percent of the recipients of cells from tumour-primed mice, cleared a challenge dose of P815 faster than mice injected with normal cells. Of these 53% survived significantly longer than control groups given either normal cells or no cells at all. Cells from mice primed to PPD showed significantly enhanced proliferative responses to soluble and P815-bound PPD, when compared with unprimed animals. However, cells from only a few PPD-primed mice showed enhanced cytotoxicity against P815 tumour cells, and PPD-primed cells either did not alter, or suppressed, the cytotoxic response of normal DBA/2 spleen cells, when stimulated with PPD-coated P815. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Tissues are heterogeneous communities of cells that work together to achieve a higher-order function. Large-scale single cell RNA-sequencing (scRNA-seq) offers an unprecedented opportunity to systematically map the transcriptional programs underlying this diversity. However, extracting biological signal from noisy, high-dimensional scRNA-seq data requires carefully designed, statistically robust methodology that makes appropriate assumptions both for the data and for the biological question of interest. This thesis explores computational approaches to finding biological signal in scRNA-seq datasets. Chapter 2 focuses on preprocessing and cell-centric approaches to downstream analysis that have become a mainstay of analytical pipelines for scRNA-seq, and includes dissections of lineage diversity in high grade glioma and in the largest neural stem cell niche in the adult mouse brain. Notably, the former study suggests that heterogeneity in high grade glioma arises from at least two distinct biological processes: aberrant neural development and mesenchymal transformation. Chapter 3 presents a flexible approach for de novo discovery of gene expression programs without an a priori structure across cells, revealing subtle properties of a spatially sampled high grade glioma that would not have been apparent with previous approaches. Chapter 4 leverages our prior work and a unique tissue resource to build a unified reference map of human T cell functional states across tissues and ages. We discover and validate a novel pan-T cell activation marker and a previously undescribed kinetic intermediate in CD4+ T cell activation. Finally, ongoing work defines key programs of gene expression in tissue-associated T cells in infants and adults and predicts their candidate regulators.

Immune exhaustion can be a major clinical problem for patients who have cancer or chronic inflammation. Persistent antigen stimulation drives T cells to express multiple surface markers called immune checkpoints. When these markers bind to their corresponding ligands that are expressed by antigen (e.g. tumor cells), T cells become metabolically impaired and lose several important functions; some cell signaling pathways are inhibited, while other intracellular mediators are re-modulated. Eventually, both CD4+ and CD8+ T cells behave dysfunctionally in ways that may facilitate cancer progression. Immune checkpoints are a major hallmark of immune exhaustion. In addition, natural killer (NK) cells, a critical immune cell subset in the peripheral immune system, also express immune checkpoint molecules, and are responsible for detecting and destroying circulating tumor cells. Yet, little research has investigated immune checkpoints on NK cells. Here, we explored the role of Bromodomain and ExtraTerminal domain (BET) proteins (BRD2, BRD3, BRD4), which are important transcriptional co-regulators, and critical for proliferation and metastasis in many cancer types, in the regulation of immune checkpoint molecules in several immune cell subsets, including CD4+ and CD8+ T cells, and NK cells. Through binding to acetylated histone tails of nucleosomal chromatin, BET proteins assist in transcription of multiple genes. Deregulated expression of BET proteins promotes cancer development or tumor cell metastasis, and new data show the BET proteins contribute to immune exhaustion. Furthermore, Type 2 diabetes mellitus (T2DM) is another worrisome problem related to cancer. T2DM patients show increased risk of developing cancer. Patients with both T2DM and any type of cancer show higher risks for metastasis. Significantly, T2DM patients also show immune exhaustion, suggesting a hypothesis that BET proteins may couple immune system dysfunction, abnormal metabolism and cancer incidence or progression. Specifically, T2DM has been defined to be a metabolic and a chronic inflammatory disease. The 5' Adenosine Monophosphate-activated Protein Kinase (AMPK) signaling pathway is a key pivot of cell metabolism and as well a significant target of drugs that normalize blood glucose, such as metformin. Based on published data, we considered that it is important to explore the mechanism of how immune checkpoints are regulated through metabolic pathways, focusing on immune exhaustion in T2DM patients. Moreover, considering that the expression of BET proteins promotes cancer development and progression, and metastasis and immune exhaustion are characteristic of many cancers as well, we suspected a potential relationship among BET proteins, the AMPK metabolic signaling pathway and immune exhaustion is worth exploring. Here, we measure expression of the immune checkpoint molecules TIM-3, TIGIT, PD-1, and CTLA-4 on normal T cells and NK cells by flow cytometry. We demonstrate different degrees of regulation of immune checkpoints by BET proteins on stimulated T cells and NK cells. Comparing stimulated-only cells with stimulated-plus AMPK inhibitor cells, we found that inhibition of the AMPK signaling pathway causes divergent expression patterns for TIM-3 and TIGIT, PD-1 and CTLA-4. Simultaneous inhibition of both BET proteins and the AMPK signaling pathway, shows that BET proteins regulate TIM-3 and TIGIT through an AMPK-independent metabolic pathway and regulate PD-1 and CTLA-4 through an AMPK-dependent pathway. Overall, we show TIM-3 and TIGIT, PD-1 and CTLA-4 display different expression patterns under regulation of the AMPK signaling pathway, and we show that BET proteins regulate TIM-3, TIGIT, PD-1 and CTLA-4 through both AMPK-dependent and -independent pathways. These findings are important because they reveal novel mechanisms of immune checkpoint regulation, which may be valuable for targeting in cancer patients who are being treated with checkpoint inhibitors.

T cells are an essential component of the adaptive immune system, which evolved to facilitate development of long-term, effective protection against infectious diseases. Upon activation, T cells play an important role in clearing infections, and especially, in preventing establishment of subsequent infections with the same pathogen. Because this is such a powerful response, it must be tightly regulated. Our lab has long been interested in how signaling molecules regulate the function of T and B lymphocytes. Our prior studies stimulated an interest in the signaling adapter molecule, Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 unexpectedly plays an important positive role in T cell functions, including providing help for B cell responses, protection from infectious pathogens, cytokine production and proliferation. After TCR engagement, TRAF3 associates with the T Cell Receptor (TCR)/CD28 complex. These data identified a new role for TRAF3 in T cell activation. There are three signals that are required for full T cell activation. The three types of receptors that deliver these signals are the TCR, co-stimulatory receptors and cytokine receptors. This dissertation explores the regulatory role of TRAF3 in the 3 signals required for T cellsactivation. In signal 1, TRAF3 enhances TCR signaling by regulating the localization of the TCR inhibitors, PTPase non-receptor type 22 (PTPN22) and the c-Src kinase (Csk). Our lab previously reported that recruitment of TRAF3 to the TCR complex requires co-stimulation of CD28, the primary receptor for signal 2. In this dissertation, we show that TRAF3 associates with the Linker of Activated T cells (LAT) complex, demonstrating preference for distinct LAT-associated proteins. For delivery of signal 3, T cells require stimulation of a cytokine receptor, such as IFNαR, for differentiation of a T cell to an effector cell. Upon IFN stimulation, TRAF3 inhibits IFNαR-induced early molecular events, which results in the regulation of both canonical and non-canonical IFNαR signaling pathways. The results presented in this dissertation highlight the dynamic roles of TRAF3 as a regulator of T cell activation, by regulating multiple T cell signaling pathways.

Adoptive T cell immunotherapy is emerging as a powerful approach to treat diseases ranging from cancer to autoimmunity. T cell therapy involves isolation, modification, and reintroduction of T cells as “living drugs” to induce a durable response. A key capability to fully realize the potential of T cell therapies is effective manipulation of ex vivo T cell activation, with the aim of increasing T cell production and promoting specific phenotypes. While initial efforts to modulate T cell activation have heavily focused on mimicking biochemical signaling and ligand-receptor interactions between T cells and antigen presenting cells (APCs), there is increasing appreciation for understanding the role of mechanics at this interface and utilizing these insights to improve T cell activation systems. The aims of this dissertation is to contribute to this understanding by elucidating how mechanical properties of an activating surface regulate T cell activation, and apply these insights to generate biomaterial based systems to enhance activation from leukemia patient derived T cells. We first use a hydrogel system to investigate patterns T cell activation to substrate stiffness, discovering a biphasic response of T cell activation to stiffness that is synergist with ligand density. We then generate electrospun fiber scaffolds as an alternative platform to improve T cell expansion; we discover that 3D geometry in the form of fiber diameter and span lengths affects T cell activation. Lastly, we characterize the starting makeup of T cell populations from leukemia patients to study patterns of T cell exhaustion, utilizing the developed electrospun fiber scaffold system to enhance expansion of exhausted T cells from leukemia patients, and demonstrate patient-specific responses to different scaffold formulations. This approach allows for engineering of biomaterial designs that can leverage T cell mechanobiology to enhance T cell activation, with potential to be tailored to patient-specific expansion conditions and increasing the availability of T cell therapy to a wider range of patients.

The Metzincins are a superfamily of zinc-dependent endopeptidases associated with the regulation of the extracellular matrix (ECM). Their members include A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTSs), A Disintegrin and Metalloproteinases (ADAMs), and the matrix metalloproteinases (MMPs). Metzincins exhibit diverse functions associated with both physiological and pathological states that include the proteolytic degradation of the ECM, regulation of various growth factors, cell surface receptors, and chemokines, and mediation of biological functions such as extravasation, survival, and proliferation. In pathological conditions such as cancer associated with chronic inflammation and multiple sclerosis associated with neurodegeneration, dysregulation of Metzincin activities are a hallmark of disease progression and severity. Hence, Metzincins are therapeutic targets for various disease states and research into optimal Metzincin inhibitor design is an ongoing exploit. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection