Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer-related deaths in the world. It is a disease with poor prognosis with unsatisfactory long-term survival of patients, and thus new strategies to control this disease are warranted. T helper (Th) 17 cells and IL-17 have recently been detected with increased frequency in a number of tumors including HCC. Its role in tumor remains controversial but its presence in HCC has been linked to disease progression, possibly involving angiogenesis. Th17 cells could be homed to inflammatory sites such as tumor microenvironment via CCR6/CCL20 axis and expand locally, and studies from other inflammatory diseases such as autoimmune disease has shown that the gut is the potential source of Th17, where its induction is affected by signals from gut microbiota. Yet this link is not yet shown in extra-intestinal tumors. Probiotics are living microorganisms, which when administered in adequate amounts confer a health benefit on the host. They have been reported to relieve chronic inflammatory diseases in animal and in human intervention studies. It is believed that probiotics regulate signals to gut antigen-presenting cells, which act as the pivot in modulating the systemic immune responses and inactivated bacteria also exhibited immunomodulatory effects in this regard. Accordingly, it was hypothesized that oral feeding of probiotics to HCCbearing animals may affect Th17 polarization and distribution and thereby modulate tumor microenvironment, which may have beneficial effect in tumor development, possibly via affecting angiogenesis. To address this hypothesis, wild-type C57BL/6 mice were fed with different heat-inactivated or viable probiotics– Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (EcN), VSL#3 or mixture of probiotics − Prohep (heat-inactivated LGG, heatinactivated VSL#3 and viable EcN) either one week in advance or at the time of subcutaneous tumor inoculation. Probiotic feeding had improved survival in tumor-bearing mice, slowed down tumor growth and reduced tumor burden when monitored for 38 days. Probiotics showed better efficacy when feeding was given in advance. The anti-tumor effect was related to reduced angiogenesis and reduced IL-17 serum and gene expression within tumor. The mechanistic link between IL-17 modulation and tumor development was further studied in animals by IL-17 neutralization. The anti-tumor efficacy of probiotics, in relation to tumor growth and angiogenesis, was lost after IL-17 neutralization, which was linked to recruitment of myeloid suppressor cells. Since cells from both adaptive and innate immune systems could secrete IL-17, the source of IL-17 production was then identified, and found that Th17 was the major IL-17 secretor being modulated by probiotic feeding. Reduced homing of Th17 to tumor via circulation, with a tendency being recruited from gut was observed. Probiotics-mediated Th17 cell modulation in the gut by inducing the skewing of IL-10 secreting type1 regulatory T cells via dendritic cells may link to limited IL-17 mediated angiogenesis in the tumor microenvironment. With better understanding of the immunomodulation properties of probiotics, prophylactic or therapeutic efficacy in management of other inflammation-associated cancer can be availed. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Diseases caused by fungi are increasing worldwide and are often associated with high mortality rates. In particular, the normally harmless commensal Candida albicans can cause serious disease if immunological and physiological barriers are perturbed, leading to systemic infection, which is fatal in up to 45% of cases. The adaptive immune response is believed to be important in protection against systemic candidiasis, however, the roles of different helper T (Th) cell subsets, particularly Foxp3+ regulatory T (Treg) cells, remain largely unexplored. The aims of this study were to adapt a mouse model of systemic C. albicans infection to test whether the numbers of Th1, Th2, Th17 and Foxp3+ Treg cells increase in mice with systemic C. albicans infection, and determine their contribution to disease. C. albicans drove the expansion of Th1, Th2 and Th17 cells, as well as multiple Foxp3+ populations that displayed characteristics of natural Treg, induced Treg, Th17 and Th1 cells in vitro and in vivo. The expanded Foxp3+ T cells inhibited Th1 and Th2, but promoted Th17, responses to C. albicans antigens in vitro and exacerbated disease, since their depletion in vivo reduced kidney fungal burden and inflammatory lesions. Furthermore, systemic infection with a weakly virulent C. albicans strain was associated with reduced Treg responses compared to those induced during lethal systemic infection. These data lead to a model for systemic candidiasis whereby Treg expansion promotes Th17 responses that drive pathology, and have implications for future immunotherapy.

The extensive infiltration of immune cells in the joints of patients affected by rheumatoid arthritis (RA), and the subsequent production of pro-inflammatory cytokines triggers bone erosion through the extensive stimulation of bone resorbing osteoclasts (OCs). The activity of γδ T cells has been implicated to influence the onset and severity of the disease pathology in murine models of human RA. With this study the effects of γδ T cells for influencing OC differentiation and resorptive activity were assessed in vitro. Activated γδ T cells exerted inhibitory effects on OC differentiation and resorptive activity, these effects were mediated by the release of soluble factors, since similar inhibitory effects were obtained using conditioned medium (CM) from activated γδ T cells. The primary mediator of such effects was determined to be IFN, since neutralisation markedly restored OC differentiation and resorptive activity. γδ T cell proliferation, activation and survival following culture with autologous mature OCs were assessed by flow cytometry. Interestingly, OCs and OC-derived CM induced activation of γδ T cells as determined by the expression of the early activation marker CD69. A mediator of this stimulatory effect on T cells was found to be TNF, since neutralisation of TNFα decreased the stimulatory effect of OCs on CD69 expression. Consistently, OCs, but not OC-derived CM, increased the proliferation of IL-2-stimulated γδ T cells and also supported the survival of resting γδ T cells. This study provides new insights into the in vitro interactions between human γδ T cells and OCs, moreover it defines osteoclasts as immune competent cells capable of influencing the activation status and the viability of T lymphocytes, and provide evidence for a novel stimulatory effect of OCs on γδ T cells.

Previous studies employing the generation of MHC-incompatible embryonic chicken chimaeras by injecting MHC-incompatible stem cells resulted in an unexpected finding. Chimaeras made late in gestation developed as adults a severe autoimmune syndrome resembling the human syndrome of Systemic Lupus Erythematosus. Work in our laboratory aims to understand the role of CD8 T cells in immunity and/or autoimmunity. We have tested a three-cell model of CD4 T cell activation and differentiation during the development of the immune response specific for MHC transplantation antigens in one way mixed lymphocyte reactions. Our model proposes that whether Th1 or Th2 immunity is generated depends on both the ratio of CD4:CD8 T cells specific for antigen at the initiation of the immune response and on the ability of antigens to coordinately induce both CD4 and CD8 T cells. Previous studies employing parent into F1 models of graft-versus-host disease in mice have shown that the injection of parental cells results in two distinct outcomes. Parental cells which do not have a sufficient number of CD8 T cells present produce an autoimmune syndrome characteristic of systemic lupus erythematosus and a chronic graft-versus-host disease mediated by a Th2 response. Conversely, the presence of an adequate number of CD8 T cells results in a Th1 immune response and acute graft-versus-host disease resulting in the death of the F1 host. Our findings indicate that the ratio of the number of CD4 T cells to the number of CD8 T cells present in the spleen is crucial in whether naive CD4 T cells differentiate into Th1 or Th2 cells. We refer to this ratio as the CD4:CD8 T cell ratio or CD4:CD8 ratio. Thus, the differentiation of naive CD4+ T cells towards a differentiated Th1 phenotype is critically dependent on the concomitant induction of CD8 T cells by the same antigen, driven by a low CD4:CD8 ratio. In contrast, inefficient induction of CD8 T cells during the initial priming of lymphocytes greatly facilitates the differentiation of CD4 T cells towards the Th2-type lineage, and occurs when the CD4:CD8 ratio is high. Given our findings on the significance of the ratio of CD4:CD8 T cells in the decision making process of CD4 differentiation stimulated by antigen, we hypothesized that different CD4: CD8 ratios at different stages of development might contribute to the immune response generated at these stages. We tested this hypothesis in mice by comparing the CD4:CD8 ratio in adults and neonates and the Th1/Th2 responses generated in vitro. This CD4:CD8 T cell ratio is significantly higher in neonates than adults resulting in predominant Th1 responses by adult spleen cells and Th1/Th2 responses by neonatal spleen cells as demonstrated by the ELISPOT assay. We have compared the CD4:CD8 T cell ratio of a large number of adult and neonatal spleens in several mouse strains and have studied it systematically in BALB/c and C57BL/6 mice by flow-cytometry. We have consistently found a 3-5 fold higher CD4:CD8 T cell ratio in neonates as compared to adults in the strains tested. Furthermore, we found that neonatal spleen cells generate a predominant Th2 response whereas adult spleen cells generate CD4 and CD8 Th1 immunity when activated under the same conditions. We have further studied the role of CD8 T cells in CD4 T cell differentiation by reconstructing the adult CD4:CD8 T cell ratio in neonatal spleen cells with age-matched, isolated CD8 T cells. We found that in these “CD4:CD8 ratio-reconstructed cultures”, the Th2/IL-4 immunity is suppressed with concomitant generation of Th1/IFN-γ immunity upon activation by allo-antigen. Additionally, we have characterized the phenotype of the T cell mediating Th1/IFNγ immunity in the “CD4:CD8 ratio reconstructed cultures” and we found that while CD8 T cells produce exclusively IFN-γ, CD4 T cells now produce IFN-γ rather than IL-4. We suggest that physiologically distinct CD4:CD8 ratios at different stages of life should be considered in designing protocols of neonatal vaccination against pathogens that are contained by Th1-type immunity upon infection as adults. Moreover, as elaborated in the discussion, our studies might be pertinent in understanding by which mechanism autoimmunity arises in some cases.

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR. To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC. Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton.

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