Minoughan, Sophie; (2021) Single molecule approaches to study the mechanics of complement receptor 2 in antigen presentation to B cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The importance of mechanical forces within the immune system has long been recognised through their roles in cell motility, cell adhesion and in the engulfment of pathogens. Additionally, mechanical forces have been found to regulate lymphocyte activation. In the case of B lymphocytes, mechanical forces promote extraction of cell-presented antigens in cell-cell contacts called immune synapses. It is hypothesised that the mechanical forces during antigen extraction promote affinity discrimination. This is required for B lymphocytes to produce high affinity antibodies and thus efficiently eradicate pathogens. This process is particularly important during presentation of antigens bound in immune complexes, which is mediated by complement receptor 2 (CR2) on follicular dendritic cells (FDCs). However, the mechanics of immune complex presentation and extraction remain poorly characterised. This thesis focuses on three main goals. Firstly, this work aims to develop an approach for measuring receptor ligand interactions under applied forces using a novel adaptation of the single molecule technique of magnetic tweezers (MT). Secondly, this thesis aims to apply the newly developed approach to study the mechanics of the interactions of CR2 with its ligand, C3dg, and with an anti-CR2 monoclonal antibody. These interactions will act as models of B cell pulling of immune complexes from FDCs. Finally, this in vitro approach is complemented by studying cellular expression of CR2 and measuring its interactions with C3dg at the surface of live cells. Through this work, I have developed a comprehensive, MT-based workflow for measuring lifetimes of single molecule interactions under externally applied forces. Although this method proved inefficient to study the fast force-mediated dissociation of single C3dg molecules from CR2, through studies of CR2 interactions with a monoclonal antibody, I have demonstrated the general applicability of this method for protein-protein interactions. I have also extended this method to use with live cells, where I found an enhanced binding of C3dg by a longer variant of CR2, CR2L, expressed on FDCs. This is the first functional difference of CR2L discovered to date and suggests that improved binding of immune complexes by FDCs via expression of CR2L may enhance B cell and antibody responses.

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