B-Cell Receptor Signalling in Chronic Lymphocytic Leukaemia.

Abstract

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.