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Title: Imaging-based analysis of antibody superagonism
Author: Assmann, Meike
ISNI:       0000 0004 6499 8578
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Activating antibodies against the T-cell costimulator CD28 fall into two categories: those that require an additional stimulus, such as an anti-TCR antibody, to fully active a T cell are termed "conventional antibodies" (CAs), and those that induce such strong signalling that T cells become activated in the absence of TCR ligation are referred to as "superagonists" (SAs). It was proposed that SAs are particularly potent because they sterically exclude large phosphatases such as CD45 more efficiently from signalling receptors versus CAs. To test this, we stimulated T-cell lines with an anti-CD28 SA or CA immobilised on glass surfaces. Although no TCR ligand is present, we confirmed that the expression of the TCR is nevertheless required for SA-induced signalling on glass. We showed that the SA is only active on glass when it is immobilised via its Fc, and that signalling is sensitive to the dimensions of the antibody/CD28 complex relative to those of CD45. We then visualised the organisation of these proteins by imaging. When treated with the SA, T cells form contacts with the glass slide that exclude CD45 ~ 2-fold more efficiently versus CA treatment, and both antibody-bound CD28 and CD45 slow down their diffusion to a greater extent under SA versus CA treatment. Both the exclusion of CD45 and the restriction of CD28 and CD45 diffusion appear to be steric effects, caused by the SA bridging a narrow gap between cell and glass surface. We hypothesize that SAs induce particularly potent signalling by protecting CD28 from dephosphorylation by CD45 via (1) spatially segregating the two proteins, and (2) reducing their encounter rate by slowing them down, while kinases continue to diffuse and have access to CD28. In summary, our results provide evidence that antibody superagonism is driven by size-dependent local perturbations in kinase and phosphatase organisation and diffusion.
Supervisor: Davis, Simon Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Immunology