Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.

Recent research of a cohort of HIV exposed sero-negative (HESN) female commercial sex workers in Nairobi has revealed an Immune Quiescent phenotype; characterized by reduced T cell activation and higher regulatory T cells (Tregs) in peripheral blood. HESN women also express lower levels of interferon regulatory factor-1 (IRF-1), a critical regulator known to negatively impact Treg development in mice. In this study, we analyzed the functional capacity of Tregs by an in vitro depletion assay and measured functionality by flow cytometry. Data showed Tregs suppressed CD4+ and CD8+ proliferation responses. We characterized the link between Tregs and IRF-1 in HESN and observed an inverse correlation between IRF-1 expression and Treg proportions. We also established reduced expression of IRF-1 in Tregs of healthy donors by flow cytometry. In a separate study, flow cytometric analysis of high-risk sex-workers revealed that CTLA-4 expression in memory CD4+cells, not Treg frequency, was associated with HIV seroconversion. / February 2017

CD8+ T-cell antigen sensitivity is critical for optimal control of persistent viral infections, including HIV-1. The devastating HIV-1 pandemic may be countered by development of a cytotoxic T lymphocyte based vaccine if qualities associated with protection can be defined at the molecular level. However, the heterogeneity of the total viral-specific CTL response confounds identification of protective correlates and T-cell sensitivity is no exception and remains controversial. To address this issue we reduced the heterogeneity of the HIV-1 CTL response to single units, generating 19 CTL clones that recognise the same HIV-1 derived epitope restricted by HLA B*08. Correlation of functional assays directly with the ability of each clone to control HIV-1 replication in vitro, the “viral suppression assay,” identified antigen sensitivity as a key quality for anti-viral efficacy. Remarkably, four clones from this panel, isolated from one individual, a long-term non-progressor, all used an identical TCR yet had distinct antigen sensitivity and suppressive activity. Two of these clones were characterised in detail, and had distinct cytokine profiles, regulated by epigenetic mechanisms, and differential expression of a group of cell surface receptors with the potential to modulate the signalling threshold to antigen. Expression of the TNFα locus of the high sensitivity clone with “Good” suppression was repressed by DNA methylation. Understanding how CTL qualities required for optimal control of HIV-1 replication differentiate and are then enriched in the total CTL response, and if repression of TNFα contributes to this process, will contribute to rational vaccine design. This is the first evidence that avidity maturation in CD8+ T cells with the same TCR affinity occurs in viral infections in humans as reported in the mouse. This suggests the induction of high sensitivity CTL will be critical for an effective HIV-1 vaccine, but offers hope that this can be achieved even in individuals without protective HLA alleles, by further exploration of peripheral avidity maturation and epigenetic regulation of the HIV-1 specific CD8+ T-cell response.

Proper T-cell activation and effector function are essential for effective immunity. T-cell antigen receptor (TCR) signals are known to regulate the outcome of differentiation, but the mechanisms remain unclear. Recent work from our lab demonstrates that the Src family protein tyrosine kinase, p56Lck, is able to specifically link TCR signals to activation of the Mitogen Activated Protein Kinase (MAPK) pathway through the function of its SH3 domain. The MAPK pathway is known to be involved in T-cell activation downstream of TCR ligation and has previously been implicated in T-helper type 2 (Th2) effector function. We have utilized an Lck SH3 mutant knock-in mouse line (Lck W97A) to investigate the potential role of this regulatory signaling mechanism in determining T-lymphocyte activation and effector function. Our results demonstrate that the Lck SH3 domain function is required for normal activation of T-lymphocytes following TCR stimulation as indicated by significantly reduced proliferation, IL-2 production, and CD69 induction in Lck W97A T-cells. Biochemical studies confirm that activation of the MAPK pathway is selectively altered in Lck W97A T-cells as P-ERK1/2 induction is significantly reduced but phospho-PLCg1 induction and calcium mobilization is unaffected. In vivo experiments demonstrate a specific and significantly impaired Th2 immunity in Lck W97A mice, with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH, or infection with the helminth Nippostrongylus brasiliensis. Th1 immunity does not appear differentially regulated in Lck W97A mice as serum levels of IgG3 and IgG2b are similar to WT following immunization with DNP-KLH, as well as serum levels of IFN-g1 following immunization with heat-killed Brucella abortus. In vitro differentiation studies confirm that Lck W97A T-lymphocytes are able to be directed to the Th2 phenotype, as indicated by intracellular staining for IL-4, with significantly increased levels of IFN-g under Th2 differentiating conditions compared to WT. These data indicate that the Lck SH3 domain regulates activation of T-lymphocytes by affecting MAPK pathway induction and demonstrate a novel and critical role for Lck in the regulation of Th2-type immunity. The Lck SH3 domain has also been implicated in the pathogenesis of Plasmodium, the causative agent of malaria. The role of the mosquito vector on malaria pathogenesis is not well understood. Initial studies examining the role of vector salivary gland proteins on cells of the innate immune system indicate that Anopheles stephensi saliva is able to enhance macrophage activation and phagocytosis as well as enhance macrophage Ag-presentation to T-lymphocytes in an in vitro model.

Hepatitis C virus (HCV) is the major cause of chronic hepatitis worldwide and a leading cause for liver transplant. Unfortunately, graft HCV infection is a universal phenomenon despite of pre-transplant prophylactic strategies. Acute HCV infection and innate immune responses elicit an inflammatory scenario that triggers the recruitment of adaptive immune response cells. Of those chronically infected, 30% experience accelerated fibrosis with concomitant cirrhosis development within 5 years post-LT and require re-transplant. With many patients responding unfavorably to antivirals and ineffective vaccines, much attention is now placed on T cell immunity in controlling HCV infection. This study represents a retrospective analysis that examined the association of T cells with respect to liver fibrosis severity progression in a prospective cohort of biopsy samples taken from 27 patients at the time of HCV recurrence disease diagnosis post-LT. For those patients, the fibrosis progression was scored 36 months post-LT by Metavir scoring system. Liver biopsies were classified based on fibrosis severity as Mild (G1; n = 12), Moderate (G2; n = 6), and Severe (G3; n = 9). Additionally, an independent set of liver biopsy samples, taken according to fibrosis severity progression, was classified (G1; n =3, G3; n = 4) and used as a validation set for CD4 gene expression. Real time PCR was performed to study the expression of immune-related genes using the Taqman® probe system. From the results analysis, the CD4 T cell marker encoding gene was down-regulated (2.9-fold) in G3 with respect to G1; although, only borderline significant (p = 0.052). This suggests an inverse relationship of CD4+T cell related-genes expression with respect to worse fibrosis progression in HCV recurrence diagnosed recipients. The validation samples showed a similar trend (1.8 fold decrease in G3 with respect to G1), although not significant. This may be due to impaired T cell function resulting from T cell exhaustion, poor dendritic cell priming and activation, or the use of immunosuppressant drugs. To conclude, CD4 could be a potential biomarker to help in identifying HCV recurrent patients with a high risk of fibrosis development soon after LT.

The cholestatic liver diseases include Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC). The aetiologies of PBC and PSC are not yet known but it is thought that there are both inherited and environmental factors associated with pathogenesis. In this thesis, I set out to explore the immunologic and virologic basis of cholestatic liver diseases. In recent years a novel human betaretrovirus has been proposed as an antigenic target in PBC. Using separate T cell assays, I analysed responses to both autoantigens and foreign antigens in parallel, backed up with a molecular approach. I have shown that although there is definite evidence of prior encounter with HBRV peptides at the immunologic level the virus is not closely associated with the pathogenesis of PBC. I have shown that there are links between the composition of the cellular infiltrate and the histologic scores as well as the clinical status. In PBC, the accumulation of CD8+ cells in particular correlates with liver fibrosis stage and Foxp3 cells may have an influence on portal and lobular inflammation. In PSC, the frequency of T helper cells and B cells correlate with alkaline phosphatase and liver stage. This may indicate a closer relationship between tissue histochemistry results and fibrosis and clinical markers in PSC than in PBC and may not simply be a result of portal tract inflammation but be associated with disease pathogenesis. The quality of life tool PBC-40 showed similar results to the original Newcastle cohort. Fatigue is the symptom with the greatest apparent impact and there was no association found between symptoms and biological parameters of disease activity and severity. In PSC, there was a negative correlation between the average CD8 count per infiltrate and the emotional and social domain suggesting alterations in CD8 liver cell counts may have an influence on human behaviour or vice versa. Overall these data provide new insight into the pathogenesis of both PBC and PSC, and provide new avenues for immunologic analysis of these diseases in future.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

The study of regulatory T cells (Tregs) is a relatively new field. Within the past few decades, research on Tregs has greatly deepened scientists’ understanding of the link between the immune system and cancer. The study of melanoma is one such cancer that has benefited greatly from this area of study. Tregs are a subset of CD4+ T cells (TCs) that are either generated in the thymus or in the periphery. The main role of Tregs in normal immune physiology is to suppress immune cells. This is an essential component in the prevention of autoimmunity. In melanoma, however, Tregs prevent components of the immune system from mounting a robust response to cancerous lesions and tumors. Tregs have been observed to infiltrate melanoma tumors due to chemokines and other soluble signaling molecules such as CCL1 and CCL22. Once Tregs accumulate inside melanoma tumors, they generate an immunosuppressive microenvironment in a contact-dependent and contact-independent manner. IL-10 secretion and use of the CTLA-4 pathway were observed to be the most well characterized modes of suppression but other mechanisms are still being discovered. Clinicians can take advantage of new therapeutics to modulate the activity of Tregs. Exogenous administration of antibodies that bind to CTLA-4, PD-1, CCR4 and other receptors and molecules can prevent Treg development and action. Preventing Tregs from carrying out their suppressive function may allow other elements of the immune system, such as CD8+ TCs, to target and destroy melanoma cells. Clinicians can also measure the relative abundance of Tregs or use the ratio of effector TCs (Teff) and Tregs to predict patient outcomes and survival. More research is needed to determine that precise mechanisms of Treg infiltration and accumulation within the tumor and the mode of Treg suppression. This paper finds that there is no standard Treg identification marker. This can lead to aberrant results and failures such as the inability to distinguish Tregs from melanoma cells that also express Treg-like markers or a failure to identify other Treg subtypes. Lack of consensus also extends to the prognostic value of Tregs due, in part, to small sample sizes and the inability to accurately identify Tregs in vivo. Future research must focus on Treg identification, action, and the elucidation of therapeutic mechanisms. These future studies will ensure that clinicians have the correct information to choose the proper melanoma treatment that will target the specific Treg populations found within patients’ melanoma tumors.

Cells have long been known to sense and respond to mechanical stimuli in their environment. In the adoptive immune system particularly, cells are highly specialized and responsible for detecting and eliminating pathogens from the body. T cell mechanosensing is a relatively new field that explores how force transmission in cell-cell interaction elicits both inter- and intra-cell signaling. Owing to recent advances in genetic manipulation of T cells, it has emerged as new tool in immunotherapy. We recently demonstrated human T cell activation in response to mechanical rigidity of surfaces presenting activating antibodies CD3 and CD28. The work in this dissertation highlights new progress in the basic science of T cell mechanosensing, and the utilization of this knowledge toward the development of a more specialized expansion platform for adoptive immunotherapies. Human T cells are known to trigger more readily on softer PDMS substrates, where Young's Modulus is less than 100 kPa as compared to surfaces of 2 MPa. While the range of effective rigidities has been established, it is important to explore local differences in substrates that may also contribute to these findings. We have isolated the rigidity-dependence of cell-cell interactions apart from material properties to optimize design for a clinical cell expansion platform. Though PDMS is a well understood biomaterial and has found extensive use in cellular engineering, a PA gel substrate model allows for rigidity to be tuned more closely across this specific range of rigidities and provides control over ligand density and orientation. These rigidity-based trends will be instrumental in adapting models of mechanobiology to describe T cell activation via the immune synapse. In what is generally accepted as the clinical gold-standard for T cell expansion, rigid (GPa) antibody-coated polystyrene beads provide an increase in the ratio of stimulating surface area-per-volume, over standard culture dishes. Herein we describe the development of a soft-material fiber-based system with particular focus on maintaining mechanical properties of PDMS to exploit rigidity-based expansion trends, investigated through atomic force microscopy. This system is designed to ease risks associated with bead-cell separation while preserving a large area-to-volume ratio. Exposing T cells to electrospun mesh of varying rigidities, fiber diameters, and mesh densities over short (3 day) and long (15 day) time periods have allowed for this system's optimization. By capitalizing on the mechanisms by which rigidity mediates cell activation, clinical cell expansion can be improved to provide greater expansion in a single growth period, direct the phenotypic makeup of expanded populations, and treat more patients faster. This technology may even reach some cell populations that are not responsive to current treatments. The aims of this work are focused to identify key material properties that drive the expansion of T cells and optimize them in the design of a rigidity-based cell expansion platform.