Embryonic stem cells (ESCs) possess hypo-immunogenic properties and have the capacity to modulate allogeneic immune response. ESCs have been shown to reduce immune activation in response to third party antigen presenting cells (APCs) in vitro and have the capacity to promote allograft survival in vivo. Clinical use of live ESCs to treat immunological disorders, however, risks teratoma or ectopic tissue formation. Accordingly, the way lab is studying the immune modulatory potentials of ESC-derived factors and recently, found that dendritic cells (DCs) treated with human ESC extracts are poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. In the present study, I found that ESC-derived extracts directly inhibit T cell proliferation and suppress their activation without inducing cell death. Furthermore, ESC extracts are able to suppress Th1 polarization while increasing the numbers of Foxp3+ CD4+ CD25+ regulatory T cells. Moreover, I found that a protein called Milk fat globule-EGF factor 8 (MFG-E8) appears to be highly expressed in ESCs. Importantly, neutralizing MFG-E8 substantially abrogated the immune suppressive effects of ESC extracts on T cell activation. These findings lead to future studies to further define specific immunomodulatory factors derived from ESCs for potential applications.

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.

No description available.

T cells are believed to be important in the control of varicella zoster virus (VZV) replication but little is known of T cell epitopes and the relationships between T cell responses, viral load and clinical disease during primary infection. I initially set to investigate the immune responses to two of the main VZV glycoproteins (gE and gI) using ex vivo and cultured IFNγ ELISpot assays. I identified several novel CD4+ T cell epitopes within gE and gI and characterized the phenotype of gE DRB1*1501 tetramer specific responses in healthy immune donors. I then set out to investigate the function and phenotype of VZV specific T cells in primary infection and their relationship to viral loads and clinical disease severity by using glycoprotein E/DRB1*1501 specific MHC class II tetramers, ex vivo IFNγ ELISpot assays and quantitative real time PCR assays. I compared the frequency and phenotype of specific T cells with virological and clinical outcomes in 32 adult individuals with primary VZV infection. In healthy immune donors, the gE specific T cells showed a early intermediate stage of differentiation with evidence of recent activation. Patients with acute primary infection had higher VZV/DRB1*1501 tetramer specific T cell responses and expressed markers of activation and effector differentiation. Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of the patients (p<0.05, r=-0.64). These data would be compatible with a role for gE and gl-specific T cells in the control of viral replication during both primary infection and re-activation.

No description available.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

The mechanism of T cell recognition is the central but unsolved puzzle of adaptive immunology. The difficulties come from the multichain structure of TCR/CD3, the binate binding structure of the pMHC molecule, the diversity of the peptides presented on the APC, the critical role of coreceptor CD4/8, the communication between TCR and coreceptor CD4/8, the complex environment of interactions taking place and the binding and signaling coupled process of recognition. Most studies were using the 3D kinetic measurements or biological functional assays to address the mechanism of the T cell recognition. However, those assays are usually either lacking of physiology relevance or missing of the initial recognition signals. Here a 2D micropipette adhesion assay with high temporal resolution (-second) was used to address the in situ kinetics of molecular interaction at the membrane of live T cells. The aim of this project is to advance our understanding to the T cell recognition mechanism. The micropipette adhesion assay was firstly used to address a simple case, the resting state pMHC-CD8 interaction. In the absence of TCR-pMHC interaction, the pMHC-CD8 interaction has a very low affinity that depends on the MHC alleles and the lipid rafts of the T cell membrane where CD8 resides, but not on the peptide complexed to the MHC and whether the CD8 is an a a homodimer or an αβ heterodimer. For cognate pMHC, following the initial observation in the F5 T cell system, the binding also displays a two-step curve in the OTI T cell system. The first-step binding occurs before one second and has a very fast on-rate and off-rate (>2s ⁻¹), and the secondstep binding follows immediately but reaches a much higher level of binding. It was identified that the first-step binding is mediated by the TCR-pMHC interaction, and the second-step binding is triggered by the TCR-pMHC interaction but mediated by CD8- pMHC binding. The two-step binding is the unique property of cognate pMHC, and it can be abolished by disrupting the lipid rafts, inhibiting the Src family protein tyrosine kinases (PTK) or protein tyrosine phosphatase (PTP). The finding of two-step binding identifies a CD8-dependent signaling amplification pathway. The data also indicated the active communication between TCR and CD8 in the antigen recognition. The crosstalk between TCR and CD8 was further dissected using two anti-CD8 antibodies 53.6.7 and CT-CD8a. 53-6.7 can significantly enhance the binding of pMHC to the T cell. Although the enhancement is directly mediated by MHC-CD8 interaction, the enhancing role of this antibody is TCR dependent. Blocking the TCR-pMHC interaction on OTI T cell or expressing CD8 alone on a hybridoma abolished the enhancement. The enhancement is also dependent on the integrity of lipid rafts and the normal function of PTP. In contrast, the antibody CT-CD8 can inhibit the binding of pMHC to the T cells and interfere with the TCR-pMHC interaction. The enhancing or inhibitory role of these two anti-CD8 antibodies is reversely correlated with the affinities of TCR-pMHC interactions. Only 53-6.7, but not CT-CD8 antibody, can phosphorylate and activate Lck. The data demonstrated a dual way crosstalk between TCR and CD8, and indicated the importance of cooperation of TCR and CD8 in antigen recognition. In the physiology condition, the TCR must accurately and efficiently recognize the cognate peptide from thousands of surrounding endogenous peptides. There is an argument regarding whether the endogenous peptides plays a role in helping the TCR recognition. Our results demonstrated that the nonstimulatory peptides can significantly enhance the T cell recognition sensitivity. In the presence of nonstimulatory peptide, the TCR can efficiently detect a single antigenic pMHC. The enhancement of recognition is due to the CD8 binding to the nonstimulatory pMHC. Blocking the CD8 binding can paralyze the enhancement. In contrast, it was found that the presence of antagonist can inhibit the binding of agonist pMHC to the T cells, and the inhibition occurs in the initial recognition step. Based on the data, an "amplification and competition" model was proposed to explain the molecular mechanism of the enhancement and inhibition function of the nonstimulatory and antagonist peptides in the T cell recognition, respectively.

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes a life-long presence in the infected host. The adaptive immune response is indispensable in controlling HCMV infection. Consequently, healthy individuals show no or mild symptoms following primary infection. In contrast, immunocompromised individuals who develop primary infection or recrudescence of HCMV can experience severe morbidity, and sometimes mortality. HCMV-specific T cell populations undergo changes in the architecture of their T cell receptor (TCR) repertoire following each episode of viral reactivation. A diverse TCR repertoire is thought to be required to provide the most efficient protection against virus infection. Perturbation to this repertoire, as can occur in immunocompromised individuals following transplantation, can lead to an increase risk of developing virus-associated clinical disease. Therefore, the study of factors influencing TCR selection is critically important in both healthy and immunocompromised individuals. To further understand the factors governing TCR selection in a persistent virus infection, the current thesis examined this process in different settings. CD8+ T cell responses to persistent viral infections are characterised by the accumulation of T cells exhibiting an oligoclonal T cell repertoire, with a parallel reduction in the naïve T cell pool. However, the precise mechanism for this phenomenon remains elusive. Here, we showed that HCMV-specific CD8+ T cells recognising distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T cell repertoire, or a private, diverse T cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public TCR was co-incident with an atypical peptide-MHC (pMHC) structure, whereby the epitope adopted a helical conformation that bulged from the peptide-binding groove, whilst a diverse TCR profile was observed in response to the epitope that formed a flatter, more ‘featureless’ landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared to the T cells expressing diverse TCR. These findings provide new insights into our understanding of how the biology of antigen presentation, in addition to the structural features of the pMHC, might shape the T cell phenotype and its corresponding repertoire architecture. Next, the role of HCMV in shaping the global and antigen-specific TCR repertoire in healthy donors was examined. First, exposure to HCMV led to an inflation of terminally differentiated CD57-expressing T cells. This effect was not seen in HCMV seronegative individuals who showed evidence of exposure to another persistent herpesvirus, Epstein-Barr virus (EBV). More importantly, these terminally differentiated CD8+ T cells in HCMV-exposed individuals displayed a highly skewed architecture of their peripheral blood T cell repertoire, with large monoclonal/oligoclonal expansions. However, ex vivo analyses of HCMV-specific T cells revealed a heterogeneous pattern of CD57 expression that showed no correlation to the antigenic source of its cognate epitope. Based on these observations, we proposed that exposure to HCMV drives the differentiation of not only the global T cell population, but select HCMV-specific T cell populations as well, and that expression of CD57 by these cells was co-incident with an oligoclonal T cell repertoire. Finally, the TCR repertoire was examined in a cohort of solid organ transplant (SOT) recipients, where primary infection or recrudescence of latent virus infection can be manifested either as asymptomatic or symptomatic disease. We examined 18 SOT recipients, and observed that symptomatic HCMV or EBV infection or recrudescence following solid organ transplantation was co-incident with a dramatic skewing of the TCR repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no HCMV/EBV infection or recrudescence showed minimal or no skewing of the TCR repertoire, and maintained TCR repertoire diversity. Interestingly, this disparate repertoire showed no correlation with levels of viral load in the peripheral blood. More importantly, we showed that large monoclonal/oligoclonal repertoire expansions was linked to the loss of antigen-specific T cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, while SOT recipients who maintained peripheral blood TCR repertoire diversity and functional antigen-specific T cell responses could resist clinical symptomatic disease in spite of high levels of viral load. Therefore, the work presented in this thesis provides additional evidence on the factors governing TCR selection in HCMV-exposed healthy individuals, as well as the consequences that perturbation to the TCR repertoire has on the functionality of the T cell compartment in immunocompromised individuals.

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.

CD4 is a co-receptor for binding of T cells to antigen-presenting cells (APC) and the primary receptor for human immunodeficiency virus-I. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cysl30 in one monomer and Cysl59 in the other one (Cys133 and Cysl62 in the mouse CD4). Thus, we hypothesized that the domain swapping of D2 in CD4 regulates its co-receptor function of antigenspecific T cell activation. We found that mild reduction of the extracellular part of human CD4 resulted in formation of disulfide-linked dimers. We then tested the functional significance of dimer formation for co-receptor function using the engineered Jurkat T cell system by expressing wild-type or disulfide-bond mutant mouse CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function as assessed by antigen-specific IL-2 production. Exogenous wild type thioredoxin, but not redox-inactive thioredoxin, could inhibit the CD4-mediated IL-2 production, suggesting that the redox state of D2 disulfide bond is controlled by this oxidoreductase. Furthermore, structural modeling of the complex ofthe T cell receptor and domain-swapped CD4 dimer bound to class II major histocompatibility complex and antigen supports the domain-swapped dimer as the immune co-receptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation isalso accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred functional co-receptor for binding to APC. Strategies to promote dimerisation of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.