During an immune response, T cell activation is mirrored by a dramatic metabolic shift from oxidative phosphorylation to glycolysis. The upregulation of glycolysis allows the cell to generate the molecules needed to rapidly proliferate and to synthesize effector molecules. The resolution of the T cell response is characterized by equally fast death of most effector T cells. The remaining T cells shift back to oxidative phosphorylation, allowing the cell to survive as a memory T cell. The upregulation of glycolysis and proliferation during the effector phase is paralleled by an increased sensitivity to T cell receptor restimulation-induced cell death (RICD). Whereas cellular metabolism and cell death are important in the proper function and response of T cells, it is not clear how metabolism regulates susceptibility to cell death, nor whether T cell proliferation and contraction are directly connected. The work presented in this dissertation provides a mechanistic link between T cell proliferation and contraction by demonstrating the regulation of caspase-3 activity by the metabolic state of T cells. In effector T cells, the cytokine interleukin (IL)-2 mediates the upregulation of glycolysis, while IL-15 induces oxidative phosphorylation and a memory-like state. IL-2 is known to sensitize T cells to RICD, while IL-15 reduces RICD and increases survival. This results from the ability of IL-2 and glycolysis to increase caspase-3 activity, whereas IL-15 induces the opposite phenotype. Activation of caspase-3 during glycolysis is mediated through clustering in lipid rafts in the plasma membrane. IL-15 is shown to inactivate caspase-3 through the posttranslational modification of protein glutathionylation, which is mediated by ROS generation in the mitochondria as a by-product of oxidative phosphorylation. We further observe that glycolysis parallels the reduced activity of the electron transport chain and oxidative phosphorylation, further increasing caspase-3 activity. This is mediated by the decreased expression of electron transport chain complexes and an increase in expression of the negative regulator of complex I, methylation-controlled J protein (MCJ). IL-15 promotes reduced expression of MCJ by its methylation. Similar to IL-15-cultured T cells, MCJ-deficient T cells manifest reduced glycolysis, caspase-3 activity, and RICD. Collectively, these findings demonstrate an adaptation that links metabolism to both cell proliferation and cell death to safeguard that proliferating cells do not escape regulation that could result in autoimmune disease or lymphomas.

Regulatory T cells (Tregs) suppress effector immune responses and have been implicated in promoting chronic viral infections. Their role during influenza infection and vaccination, however, is still unclear. Influenza is a major public health concern, claiming over 49,000 lives annually in the U.S. alone. Vaccination is the best approach for preventing disease but frequent mutations of immunogenic epitopes requires a new vaccine to be formulated and administered annually. This poses a challenge for vaccine manufacturing and may strain patient compliance. A universal influenza vaccine, which targets the highly conserved extracellular domain of the influenza matrix protein 2 (M2e), may circumvent this problem by generating cross-protective immunity. In this study, we tested the efficacy of the M2e universal vaccine in the ferret, and determined whether vaccination induces a Treg response after influenza infection. We found that vaccination promotes the development of M2e specific IgM and IgG antibodies after boosting. Upon challenge with A/Memphis H1N1, vaccinated ferrets exhibited a lower body temperature and reduced virus titer compared to non-vaccinated animals. Together these findings suggest that the M2e vaccine protects ferrets against influenza infection. In order to determine whether Tregs increase after vaccination in ferrets, we had to first clone and characterize genes involved with Treg phenotype and function including CD25, Foxp3, and IL-10. The reciprocal nature between Tregs and Th17 cells and their involvement during influenza infection prompted us to also clone ferret IL-17F. Using these sequences, we designed a qRT-PCR array to measure the expression of Foxp3, IL-10, and IL-17F in ferret tissue. We also identified cross-reactive antibodies against ferret CD8, CD25, and Foxp3 for use in FACS, western blot, and ICC. Using these tools, we found that vaccination significantly increased the expression of Foxp3 in the spleen. An increased percentage of Foxp3+ lymphocytes was detected in both the PBMCs and splenocytes of immunized animals. In contrast, IL-10 and IL-17F expression decreased significantly in both immunized and non-immunized ferrets compared with naïve animals. These studies suggest that the M2e influenza vaccine induces a regulatory T cell response in ferrets and protects against influenza infection. / acase@tulane.edu

We have recently demonstrated in a model of influenza A virus (IAV) infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDC) and CD8a+ DC, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells. Reconstitution of the lungs with physiologic numbers of pDC or CD8a+ DC is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent upon direct DC:T cell interactions, DC-expressed MHC I and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DC or alveolar macrophages was unable to rescue the virus-specific CD8 T cell response. Following IAV infection there is an abundance of IAV antigen and MHC I expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8a+ DC-mediated rescue of the T cell response in the lungs. Further, although it was known that the reduction in virus-specific CD8 T cells in the lungs was a result of increased T cell apoptosis, it remained unclear what pathways of apoptosis were contributing to the increased cell death, and what mechanism pulmonary DC subsets were utilizing to rescue this defect. Here, we demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis via both extrinsic activation induced cell death and intrinsic activated cell-autonomous death pathways. Reconstitution of aDC depleted lungs with pulmonary pDC and CD8a+ DC promotes increased T cell expression of the pro-survival molecule Bcl-2 and hence, increased T cell survival and accumulation in the lungs. Our studies herein demonstrate that pulmonary DC subsets utilize a variety of mechanisms to promote the rescue of virus-specific CD8 T cells in the lungs. Blockade of the costimulatory molecules CD70, and in some cases, 4-1BBL and OX40L, ablates the pulmonary DC mediated rescue of CD8 T cell numbers in the lungs, suggesting that late costimulation is one essential mechanism that pulmonary DC use to regulate CD8 T cell immunity following IAV infection. Further, we demonstrate that the absence of DC following IAV infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC-mediated rescue of virus-specific CD8 T cell responses in the lungs requires the trans-presentation of IL-15 via DC-expressed IL-15Ra. In addition to the role of pulmonary DC mediated costimulation and IL-15 trans-presentation, we further demonstrate a previously unrecognized role for viral antigen in regulating the accumulation of both pulmonary DC and virus-specific CD8 T cells in the lungs, suggesting that viral load can dictate the nature of the inflammatory environment in the lungs and thus, regulate the character of the ensuing IAV-specific immune response. Collectively, the results detailed here demonstrate a previously unrecognized role for pulmonary DC in regulating primary IAV-specific CD8 T cell immunity, and hence, promoting enhanced viral clearance and recovery from disease.

An imbalance of the T cell immune response is observed in inflammatory bowel disease. Intestinal microbes have been linked to the disease and the disease process leads to severe mucosal injury and systemic translocation of bacterial products. Aminosalicylates, corticosteroids and immunomodulators reduce these aggressive activities but are associated with potentially serious adverse events. The aim of this work was to investigate the effects of administration of prebiotics and probiotics that modulate the gut microflora and modulate the immune response, in ameliorating severity of colitis. The prebiotic, high amylose maize resistant starch was used at two different concentrations. A number of Bifidobacterium and Lactobacillus strains were used as probiotics. BALB/c mice were administered the prebiotics and probiotics and intrarectally infused with 2.5 mg trinitrobenzene sulfonic acid (TNBS) in 45% ethanol, thereby generating colitis. Mucosal cytokine responses, colonic microbial profiles and disease activity indices were monitored. The 5% concentration of high amylose maize resistant starch delayed progression of TNBS colitis as evidenced by reduced weight loss, lesser tissue damage, abrogation of the expression and synthesis of IFN-?? and upregulation of IL-4 and IL-10. The 30% concentration of high amylose maize resistant starch exacerbated the inflammatory response with an increase in acetic acid, coliforms and endopores in the colonic contents. Three strains of bifidobacteria and 3 strains of lactobacilli were individually screened for their activity against TNBS colitis. Each strain had a distinctive effect on the course of colon inflammation. Lactobacillus fermentum VRI 003 was selected for further study as it provided most protection. The ratio of immunosuppressive cytokines to pro-inflammatory cytokines was restored closer to the normal T cell cytokine levels. It also reduced the incidence of translocation of enteric bacteria into the spleens. Dosing a minimum daily dose of 6x109 CFU L. fermentum VRI-003 to ulcerative colitis patients in remission and maintained on standard therapy for 6 months prevented the exacerbation of symptoms, including diarrhea and abdominal pain, and improved the patient general well being. It also suppressed production of IFN-?? and sustained IL-10 levels. Moreover, absence of endospores and lower numbers of coliforms were detected in the faeces of UC patients during L. fermentum VRI-003 treatment. In summary, 5% high amylose maize resistant starch and L. fermentum VRI 003 prevented colon inflammation by changing the nature of the T cell immune response and modifying the colonic microflora in the murine model. The clinical evidence supported these findings.

Abstract not available

In Pseudomonas aeruginosa, which causes severe secondary infections in immunocompromised patients, virulence factor expression is regulated by quorum sensing signal molecules known as acyl homoserine lactones (AHLs). One of the major AHLs produced by P. aeruginosa, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), has also been shown to alter the function of a range of mammalian cells. The goals of experiments reported in this thesis were to use murine models to investigate the effects of in vivo exposure to OdDHL on TH responses, define the direct effects of OdDHL on TH cells and to explore the mechanism by which OdDHL alters the function of TH cells. It was found that in vivo exposure to OdDHL led to changes in cytokine and antibody subclass production indicative of a shift towards the underlying TH bias of the mouse strain studied. Such shifts may play a role in infections with P. aeruginosa, as strong TH1 or TH2 responses have been associated with worsening prognosis for the host, while more balanced responses have been associated with decreases in both infection and pathology. These results suggest that treatments targeting the immunomodulatory activities of OdDHL may be of benefit in the clinical setting in the future. Direct analysis of TH cells in defined in vitro systems revealed that exposure to OdDHL led to uniform decreases in cytokine production and proliferation. These decreases in cytokine production were found to be the result of OdDHL acting on both TH cells and the antigen presenting cells (APCs) that activate them, and only occurred when cells were exposed to OdDHL within 4 hours of stimulation. These findings suggest that OdDHL is acting on a molecular target common to several cells types, and that in TH cells and APCs, this target is involved in the early stages of TH cell activation. Experiments in which T cells were activated with mitogens that bypass the cell membrane revealed that OdDHL is not acting on the cell membrane or membrane-associated activation factors, suggesting that OdDHL is instead inhibiting TH cell function through interactions with one or more intracellular signalling molecules.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

The work presented here outlines experiments done using a novel RF exposure chamber. This device allows biological cells to be exposed to microwave radiation similar to those emitted by mobile telephones, whilst imaging them using a laserscanning confocal microscope. Jurkat E6.1 T lymphocytes in the exposure chamber were kept within �0.5�C of 37�C, allowing for the investigation of possible athermal effects of microwave energies. These cells were loaded with the fluorescent probe Fluo-3 AM, which is specific for calcium ions, and were monitored over two 10minute periods. The first period being a control period, the second being a period where the cells were either exposed to RF energy or sham exposed. Another 5min imaging period was for the positive control,where maximal fluorescence can be achieved by the addition of the ionophore A23187. 5 different conditions for cell exposure were investigated. Both continuous wave 900MHz and continuous wave 900MHz pulse modulated at 217Hz exposures were carried out on cells that were either unactivated, or those that were activated by the mitogen phytohaemaglutinin (PHA). For these 4 conditions the average Specific Absorption Rate (SAR) was calculated to be 1.5W/kg. A group of unactivated cells were also exposed to continuous wave 900MHz energy with an average SAR calculation of 7.5W/kg. Results showed that no significant changes in calcium ion levels occurred when averaged fluorescence slopes were compared between RF exposed cells and the control period. The mean change in slopes exposed/sham period � control period)between cells that were exposed and those sham exposed also showed no significant difference. Following an inference made in the work of Galvanovskis et al. (1999)1 who showed there is a change in the calcium ion oscillation spectrum as a result of 50Hz magnetic fields, a measure of the mean frequencies of all cells was determined using a Fast Fourier Transform (FFT) analysis. The change in the average mean frequencies in cells was then measured for all conditions. Of statistical significance was the change in average mean frequency between the control period and the sham/exposed period between cells that were exposed and those sham exposed, when cells were activated by PHA. The results also showed that there was an overall drop in average mean frequency as a result of RF exposure. Assuming there is a biological significance to the findings of this thesis, careful analysis of the calcium dynamics of tissue samples and cell types associated with RF exposure from mobile phones would need to be carried out to determine what they are. This was unfortunately beyond the scope of the present study.

Bibliography: leaves 250-284. / xviii, 284 leaves : ill., plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, 2004

Amendments appended. / Bibliography: leaves 140-165. / xi, 165 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 2005