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

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

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2013

Author dc:creator

• Mohan, Bindu

Advisor dc:contributor.advisor

• Crocker, Paul

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/93d42a43-7c3c-4d6e-b69a-27cb4673c1db

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/93d42a43-7c3c-4d6e-b69a-27cb4673c1db