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

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.