Role of Siglec-7 in ganglioside recognition and modulating NK cell biology

  • Bindu Mohan

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Sialic acid binding Ig-like lectin-7 (Siglec-7), expressed primarily on NK cells,
    binds preferentially to alpha2,8 linked disialic acids such as present in the
    ganglioside GD3 that is upregulated in certain cancers. Siglec-7 is classified as
    an inhibitory receptor as it contains immunoreceptor tyrosine based inhibitory
    motifs. It has been shown to inhibit NK cytotoxicity in cellular assays thereby
    implying a role for it in NK cell mediated tumour surveillance.
    The aim of this project was to study factors affecting ligand recognition by
    Siglec-7 and its impact on NK cell functions. An investigation into the
    mechanism by which Siglec-7 mediates inhibitory signals to regulate NK cell
    biology was also carried out. Recognition of Siglec-7 for GD3 has been reported
    to be altered in the presence of complex gangliosides. This project was initiated
    with an aim to examine the role of such cis-interactions between GD3 and other
    gangliosides such as GM1 in biological systems and thereby its impact on NK
    cell biology. B16 (78) cell line was genetically modified to over-express both
    GD3 and GM1.
    This model system was then analysed using Siglec-7-Fc precomplexes for the
    recognition of GD3. Siglec-7-Fc binding of B16 (78) cells with high expression
    of GD3 and GM1 was significantly lower compared to cells having high
    expression of GD3 and low expression of GM1. However further investigation
    of these cis-interactions by confocal microscopy revealed that only less than 3%
    of the cells had patches of co-localization of the two gangliosides. Such lateral
    segregation of co-expressed GD3 and GM1 was also observed in another cell
    line model. Next, an investigation into the role of GD3 in modulating NK cell
    functions via Siglec-7 was carried out. Primary PBMCs and a Siglec-7 deficient
    NK cell line, NK92, were used for this purpose. The data obtained showed that
    Siglec-7 could negatively modulate NK cytotoxicity towards targets expressing
    disialylated ligands such as GD3. Furthermore Siglec-7 was also able to
    modulate integrin functions on NK cells. LFA-1 mediated adhesion of effectors
    to ICAM-1-Fc coated plates and the polarization of perforin granules to ICAM-1-
    Fc coated beads were negatively affected by the expression of Siglec-7 in the
    NK92 cell line. Biochemical analysis of LFA-1 mediated signalling in NK92 cells
    showed negative regulation of Src kinase activation, in an Siglec-7 dependent
    manner. Overall these findings suggest a role for Siglec-7 in modulating NK cell
    recognition of tumours with aberrant glycosylation patterns. They also form the
    basis of further investigation into the mechanisms of inhibitory signalling
    mediated by Siglec-7 and could therefore be of potential clinical relevance in NK
    cell mediated tumour clearance.

    Date of Award2013
    LanguageEnglish
    Awarding Institution
    • University of Dundee
    SupervisorPaul Crocker (Supervisor)

    Cite this

    Role of Siglec-7 in ganglioside recognition and modulating NK cell biology
    Mohan, B. (Author). 2013

    Student thesis: Doctoral ThesisDoctor of Philosophy