Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliographical references.

We used Experimental Autoimmune Uveoretinitis (EAU) as a model system to investigate the involvement of CCR2 and CX₃CR1 in regulating the trafficking and function of monocytes and microglia in an autoimmune context. METHODS: W.T. or CX₃CR1^GFP/GFPmonocytes were adoptively transferred into mice with EAU.  At 48 hours post transfer their phenotype was examined by flow cytometry and monocytes trafficking to the retina was imaged using Scanning Laser Ophthalmoscopy. An anti-CCR2 antibody (MC21) or antagonist (JE(9-76)) was used to examine the effect of CCR2 blockade on W.T. monocytes trafficking.  Infiltration of monocytes into the inflamed retina and activation of retinal microglia were examined by confocal microscopy on retinal flatmounts from W.T. and CX₃CR1^GFP/GFP mice and immunohistochemistry on cryosections from eyes. RESULTS: CCR2 increased on W.T. monocytes at 48 hours post transfer and at 24 hours on CX₃CR1^GFP/GFP monocytes.  However, blocking CCR2 by either method did not reduce the number of W.T. monocytes rolling along retinal vessels and infiltrating the retina.  Lack of CX₃CR1 did not alter microglial activation but infiltrating monocytes lacking the receptor could not migrate through the retina and clustered around vessels. CONCLSIONS: CCR2 may  not always be needed for recruitment into an inflammatory site.  CX₃CR1 has a role in neuroprotection in the retina by enhancing the migratory ability and distribution of infiltrating monocytes within inflamed tissue.  This work stresses the importance for careful dissection of the chemokine receptors’ mechanism of action before therapeutic possibilities are explored.

Dendritic cells play an essential role in activating immune responses upon recognition of pathogens. This results in maturation and migration to the lymph nodes, where T cells are stimulated by upregulated antigen presentation, co-stimulation and cytokine secretion. DCs are also considered important in inhibiting inappropriate immune responses against self-peptides which could lead to the development of autoimmunity. This has been attributed to DCs that demonstrate inhibited co-stimulation and cytokine secretion. It has been previously shown that the continuous ligation of an immunomodulatory receptor, LILRB1, during DC differentiation results in such a DC population that demonstrates an immature phenotype even after exposure to bacterial components and resulted in inhibiting primary T cell responses. The mechanisms by which LILRB1-DCs promote tolerance are, therefore, here investigated. Previous studies revealed significantly altered expression for a large number of gene targets which varied from immune to cytoskeletal and bone-related functions. One of these includes DcR3, a soluble protein with a poorly defined role in immune regulation. It is here demonstrated that DcR3 has a positive role in the induction of IL-17, a cytokine implicated in autoimmunity. However, DcR3 was not secreted by LILRB1-DCs, possibly accounting for some of their tolerogenic functions. In addition, the expression of several cytoskeletal proteins was significantly changed in response to LILRB1 ligation and was associated with decreased ability for phagocytosis and migration. Lastly, it has been recently identified that DCs are able to trans-differentiate into osteoclasts, the main cell type linked with inflammatory bone disorders, such as rheumatoid arthritis. It is here shown for the first time that ligation of LILRB1 inhibits this process and results in decreased bone resorption. Overall, these data provides evidence that ligation of LILRB1 on DCs affects normal inflammatory functions and suggests its potential for the development of new treatments against several autoimmune diseases.

Chronic Lymphocytic Leukaemia (CLL) remains the most abundant leukaemia in those aged over 65 years. It is characterised by the expansion of malignant monoclonal B-lymphocytes that were originally described as being functionally incompetent. Identified by immunophenotypic expression of monoclonal light chain restriction, it falls into the classification of chronic B-cell lymphoproliferative disorders (B-LPD). This thesis aims to demonstrate that CD160, an activating NK cell receptor, is aberrantly expressed in B-LPD and can function as a tumour specific antigen, which has clear translation roles within the clinical environment, aiding in the diagnosis of CLL and monitoring of minimal residual disease (MRD). More so, this study aims to provide an insight into the potential biological roles of CD160 within chronic B-cell malignancies. CD160 is an activating NK cell receptor whose major form is a glycosylphosphatidylinositol (GPI)-anchored cell surface molecule with a single immunoglobulin domain. In-vitro studies on a large cohort of B-LPD patients demonstrated that CD160 was primarily restricted to cases of CLL (98%) and Hairy Cell Leukaemia (HCL, 100%) with only a minor population of other B-LPDs expressing the antigen. More so, within the B-cell lineage, CD160 can be considered a tumour specific antigen (TSA) in that when looking for both transcript and protein, they were absent throughout the normal B-cell hierarchy. Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. Disease eradication has been shown to be prognostic. This study demonstrates the feasibility and clinical importance of MRD detection utilising CD160 as novel marker of residual disease. Subsequently, CD160 analysis by flow cytometry (CD160FCA) demonstrated to be as sensitive and specific as other methodologies, and independent of the type of therapy. Further to this the early detection of MRD was correlated with known biological prognostic risk groups. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and M), the time to detection of MRD or relapsing disease ß2using CD160FCA, was significantly shorter than those with a normal or good prognosis. Within normal NK and T lymphocytes, CD160 has a multifunctional role that upon triggering results in a unique profile of cytokine production via the recruitment of Phosphatidylinositol 3-kinase (PI3K). In CLL cells, CD160 stimulation resulted in the recapitulation of these observations including cell survival, an increase in Bcl-2 family antiapoptotic proteins, and cell cycle progression. This thesis has demonstrated that CD160 is aberrantly expressed in malignant B-cells, it has a clear clinical translation role in terms of diagnosis and MRD monitoring, and multiple biological functions which recapitulate those observed in NK-cells.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 215-232). Also available in electronic version.

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

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Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 91-106) Also available in print.