Bias-free imaging of antigen degradation using bioorthogonal antigens

Abstract

Abstract Degradation of antigen into short MHC-loadable polypeptides by antigen presenting cells presents a Heisenbergian conundrum: reporter strategies for imaging these processes affect the processes itself to such an extent that the information obtained from reporter antigen strategies may not be representative of the process itself. For example fusion constructs of antigens are only imagable until they are degraded or separated from the antigen, which is essential if a peptide from the antigen is to be loaded on an MHC and reach the surface for sampling by T-cells. This results in an imaging bias to only those parts of the pathway in which reporters remain intact. Further complications can arise from the fact that reporter-constructs can alter the rate of proteolysis and routing by virtue of changing overall physiochemical properties of the antigens. I will present our recent developments of ‘bioorthgonal antigens’, a bias free antigen labelling strategy that allows the imaging of antigen routing with minimal interference in the pathway. These antigens carrying amino acids with tiny chemical modifications (2 or 3 atoms in size) in their side-chains that are stable to proteolysis and other conditions found in the cell, can be selectively visualised using click chemistry. This allows, for example the localization of degrading antigen inside an APC or the determination of the rate of epitope appearance on the cell surface without the need for peptide-MHC-specific antibodies. This offers the first opportunity to have a detectable group that does not affect processing and presentation