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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.

Chronic Lymphocytic Leukaemia (CLL) cells may depend on B-cell receptor (BCR) signalling as well as other microenvironmental survival signals. A peculiarity of CLL is that cells preserve IgD signalling in the presence of reduced IgM signalling, a pattern mimicking anergic B-cells, and consistent with autoantigen exposure. This aim of this thesis was to examine the differing roles of IgM and IgD in CLL. IgM and IgD expression was examined in CLL cells from peripheral blood (PB) and lymph node (LN). Co-expression of IgM and IgD was common, but levels of expression of IgD and IgM vary independently within these compartments, implying they may have differing roles. Most PB CLL samples underwent calcium (Ca) flux after IgD ligation, with evidence of relative IgM anergy. Incubation of cells for 24h in vitro partially restored IgM Ca flux, further supporting an anergic model. A pro-survival role of the BCR was suggested by the finding that BCR ligation was associated with reduced apoptosis in vitro. Mechanistic differences of IgM and IgD signalling were examined using mass-spectrometry based phosphoproteomics. Six CLL samples were compared to five tonsil controls, and >5000 unique phosphopeptides identified. CLL and tonsil phosphoproteomes were compared. BCR induced changes in phosphoproteins and kinases differed between IgM and IgD ligation, and between CLL and tonsillar B-cells. Recognised and novel pathways included BCR, spliceosome and Notch signalling. Evidence in support of anergic signalling in CLL was observed. Anergic IgM signalling is contrasted with IgD as a dynamic process, different in the tissue compartments of CLL. Phosphoproteomics offers a powerful tool for interrogating intracellular signalling, with phosphorylation networks characterizing pathway topology. BCR signalling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known as well as novel pathways that may well represent novel treatment targets.