Use of proteomics to study Fc gamma RIIB

Abstract

Fc gamma receptors (FcγR) bind to IgG antibodies and regulate their biology. FcγRIIB is the sole inhibitory FcγR in humans and mediates both normal immune responses and antibodymediated immunotherapy. In order to better understand and manipulate this critical receptor, it is essential to have understanding of FcγRIIB engagement, downstream signalling and regulation. The work in this thesis describes a series of proteomics experiments designed to address this gap in knowledge. These techniques, employing liquid chromatography based mass spectrometry, allow the non-targeted relative quantification of thousands of proteins and a direct comparison between multiple samples within the same experiment.

Firstly, Ramos cell lines expressing either FcγRIIB1 or FcγRIIB2 proteoform of FcγRIIB were stimulated with immune complex or an anti-FcγRIIB agonistic antibody. Secondly, the response of primary human monocytes expressing high levels of FcγRIIB following high density (HD) culture was assessed after these stimulations. Stimulation of Ramos cells and primary monocytes was analysed, identifying proteins from several pathways, including FcγRIIB signalling and clathrin mediated endocytosis, as significantly different, indicating a clear difference in protein expression after stimulation of different proteoforms of FcγRIIB.

Subsequently, the mechanism of the FcγRIIB upregulation on monocytes after HD culture was explored, revealing a distinct phenotype including proteins and pathways mapping to hypoxia, glycolysis, adhesion, complement and HLA class II. Changes in the surface expression of many of these proteins was confirmed by flow cytometry and proteins were often associated with open chromatin modifications as detected by ATAC-seq. Finally, similarities were identified in the phenotypes of HD monocytes and monocytes stimulated with DMOG, a pharmacological regulator of the key hypoxia regulator HIF; indicating that Page | II hypoxia is a central component of the FcγRIIB upregulation. Furthermore, these studies identified a potential transcriptional network underpinning the mechanism of FcγRIIB upregulation. Together these studies identify a series of protein changes, signalling networks and potential mechanisms associated with FcγRIIB upregulation which extend our understanding of this key protein and provide the framework for subsequent therapeutic intervention.

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Identifiers

ORCID for Sofia Anna Macari: orcid.org/0000-0002-6506-4051

ORCID for Spiros Garbis: orcid.org/0000-0002-1050-0805

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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