Mice with human immune systems, generated by transplanting human CD34+ cells into immunodeficient mice, are essential tools for studying phenomena unique to the human immune system or poorly reproduced in existing mouse models. Human immune tolerance induction, function and autoimmunity have been poorly modeled in conventional murine models, which often have poor predictive value for preclinical development. Models that allow the study of human immune cells with the reproducibility and flexibility of small animal models are required. In our lab, humanized mouse models have been used to study preclinical protocols for human xenotolerance induction and to better understand the immunological underpinnings of human autoimmunity. These are each areas of critical unmet medical need. Xenotolerance-inducing protocols may be necessary to allow long-term survival of a transplanted pig organ in a human patient, and, with more than 113,000 Americans currently waiting for a life-saving organ, the need to expand the pool available for transplantation is urgent. Additionally, clinical options for patients with autoimmune diseases are limited. Currently, most patients with autoimmunity are only diagnosed after significant immune damage of target organs. Predicting who will develop autoimmunity – and who will not – before damage occurs would be very useful but is currently very difficult. Small animal models that can better help us understand how human autoimmunity develops could help us develop protocols for early detection and even prevention. We have developed a personalized immune model to study the development of an individual patient’s immune system in a transplanted mice to better understand immune abnormalities that underlie autoimmunity. We have used existing humanized mouse models to answer important questions related to human xenotolerance induction and autoimmunity, but in the studies described here we have worked to extend our capacity to use these models to study human T cell development and peripheral function. We would like to be able to study both the initial selection of T cell receptors (TCRs) in the thymus based on their ability to recognize antigen in the context of presenting MHC without reacting unduly to self-antigen, as well as in the periphery, where T cells interact with peripheral antigen-presenting cells (APCs) to maintain homeostasis and respond to antigen. First, we have incorporated TCR transgenesis into our humanized mouse models to allow greater precision in studying thymic selection in our humanized mice. Developing a system for this would allow us to study in greater detail mechanisms of human xenotolerance induction, including confirming that a swine thymus can support positive selection of T cells with human-restricted TCRs to allow a future xenotransplantation patient to maintain immune competence, while also robustly tolerizing human T cells expressing pig-reactive TCRs. We will also expand this system to study the thymic selection of human T cells with autoreactive TCRs to better understand mechanisms of central tolerance and understand how they fail in autoimmunity. Finally, while processes of thymic selection are critical for human T cell development and function, peripheral interactions also have a large impact on human T cell function and homeostasis and may contribute to the development of autoimmunity. For these interactions to occur appropriately requires robust engraftment and reconstitution of APCs, especially of myeloid and B cell lineages, in transplanted immunodeficient mice. APC reconstitution tends to be suboptimal in humanized mice and is even more so in mice transplanted with patient-derived CD34+ cells. Better characterization of human APC populations and their progenitors could allow us to develop approaches to improve long-term human APC reconstitution in patient-derived humanized mice, allowing us to more fully model patient peripheral T cell function.

Chronic Kidney Disease (CKD) is a progressive condition that in the majority of cases leads to End Stage Renal Failure (ESRD) and the need for dialysis, with the only cure being renal transplant. CKD affects both adults and children; however the underlying causes of the disease are different. CKD in adults is most commonly secondary to diabetes and/or hypertension while CKD in children is usually caused by congenital structural abnormalities that result directly in renal dysfunction. There have been numerous reports of inflammatory and immunological disturbances in adult CKD that involve both the cellular and humoral immune systems. Consequences of these include an increased rate of cardiovascular disease (CVD), decreased response to vaccinations, as well as increased rates of infection, anaemia and malnutrition. Children with CKD display many of the clinical complications seen in adult kidney disease that are associated with inflammatory and immunological changes. In adults however, many of the primary conditions associated with CKD are inherently pro-inflammatory; therefore it is not clear whether the inflammatory changes observed in adults with CKD are due to pre-existing inflammatory conditions, renal disease per se or a combination of both. The majority of CKD in children is caused by conditions that are not inflammatory in nature. This presents a unique opportunity to study the inflammatory consequences of CKD alone, without the added complication of underlying inflammatory disorders. Despite this, there has been little investigation of the inflammatory and immunological status of children with CKD. Some very recent studies have shown that children with CKD have an increased systemic inflammatory state[1-3], however the nature of these immunological and inflammatory changes remains poorly defined. Identification of the specific inflammatory processes that occur in CKD may provide new treatment targets and the opportunity to develop urgently needed new therapies. The purpose of this thesis is to investigate the presence of immunological changes associated with inflammation in children with CKD. This is the first study to include children with very mild disease, and the significant changes that are present in the early stages of the disease are of particular note. I have shown that CKD in children is an intrinsically inflammatory condition, with increased accumulation of markers of oxidative stress and production of pro-inflammatory cytokines. The inflammatory markers identified in this study may be applied as a foundation for more sensitive diagnostic markers of disease progression as well as provide a basis for novel treatment strategies in this group of patients. Early identification of increased inflammation is a prerequisite for the application of preventive strategies. In addition, a better understanding of the level and mechanisms of systemic inflammation in children with CKD may enable a more accurate assessment of their risk of other inflammatory conditions such as CVD, anaemia, muscle wasting, and malnutrition. Future research that specifically focuses on the reasons and mechanisms for different rates of disease progression may emerge as a result of this study. Importantly, the findings of this study may have implications in the long term treatment of disease and may allow identification of new treatment strategies to achieve better patient outcomes. The outcomes of the study are: • Better definition of inflammatory profiles in paediatric CKD and correlation with disease severity and progression, which should contribute to improved management strategies. • Identification of new treatment targets to reduce the damage caused by chronic systemic inflammation. • Mechanistic understanding of the relationship of the inflammatory profile in regard to source leucocytes or other contributing cell types. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330366 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

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Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 110-117).

The involvement of regulatory T cells (Tregs) in immune homeostasis is now recognized as one of the fundamental mechanisms of immune tolerance. While several different types of Tregs cooperate to establish and maintain immune homeostasis, much current research is focused on defining the characteristics of the CD4⁺CD25⁺ Treg subset, as these cells can mediate dominant, long-lasting and transferable tolerance in many experimental models. The aim of this research was to characterize the biological role of a protein known as forkhead box P3 (FOXP3) that was initially identified as an essential transcription factor for the development of mouse CD4⁺CD25⁺ Tregs, in human CD4⁺ T cells. Following confirmation that, like mouse Tregs, human Tregs also expressed high levels of FOXP3, several approaches were used to investigate the role of this protein in human CD4⁺ T cells. 1) Characterization of endogenous FOXP3 expression in CD4⁺ T cell subsets revealed that this protein is not a Treg-specific marker as was previously thought. Instead, low-level and transient expression was found to be typical of highly activated non-regulatory effector T cells. 2) To generate large numbers of Tregs suitable for cellular therapy, the capacity of ectopic FOXP3 expression to drive Treg generation in vitro was explored. It was found that high and constitutive expression mediated by a lentiviral vector, but not fluctuating expression driven by a retroviral vector, was sufficient to generate suppressive cells. Over-expression strategies were also used to characterize a novel splice isoform unique to human cells, FOXP3Δ2 (FOXP3b). 3) To further probe the requirements of FOXP3 to induce suppressor function, a system for conditionally-active FOXP3 ectopic expression was developed. These studies established that FOXP3 acts a quantitative regulator rather than a “master switch” for Tregs, and that there is a temporal component to its capacity to direct Treg phenotype and function. In summary, this research has significantly expanded the understanding of the biological function of FOXP3 in human CD4⁺ T cells. Based on the potential of these cells to be manipulated for therapy, this work contributes to the field of immunology on both academic and clinical research fronts.

The goal of cancer immunotherapies is to boost the immune system's ability to detect tumor antigens and mount an effective anti-tumor immune response. Currently, adoptive T cell transfer therapy (ACT), the administration of ex vivo expanded autologous tumor-specific T cells, is one of the most promising immunotherapies under development; however, its efficacy has been limited so far with a mere 10% complete remission rate in the most successful clinical trials. The prolonged ex vivo culture process is a potential reason for this ineffectiveness because the transfused cells may reach replicative senescence and immunosenescence prior to patient transfer. The objective of this thesis is to offer two approaches towards an improvement of treatment efficacy. First, we generated a 'senescence metric' from the identification of biomarkers that can be used in the clinic towards predicting age and responsiveness of ex vivo expanded T cells. The second approach is to understand at the molecular level the changes that occur during ex vivo expansion to devise improved ACT protocols. In particular, we focused on the shift towards a pro-oxidizing environment and its potential effects on calcium signaling. The combined development and application of microfluidic technologies and computational models in this thesis facilitated our investigations of the phenotypic and signaling changes occurring in T cells during the progression towards immunosenescence. Our findings of altered T cell properties over long term culture provide insight for the design of future cancer immunotherapy protocols.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.