Bioorthogonal Antigens Allow The Study Of Intracellular Processing And Presentation Of Post-Translationally Modified Antigens

Abstract

There is a lack of tools to image and assess the impact of post translational modifications (PTMs) on antigen processing and presentation in antigen presenting cells (APCs). The existing toolbox mainly consists of fluorophore pre-conjugated peptides and proteins, which suffer from potential alterations imposed on the biological processes by the hydrophobic fluorophores. In this work, we have employed bioorthogonal chemistry in which biologically inert chemical handles are introduced into peptide or protein antigens followed by subsequent ligation of a fluorophore to the chemical handle via coppercatalyzed-azide-alkyne-cycloaddition (CuAAC). By performing this fluorophore ligation reaction after uptake and processing in the APC, the study of individual effects of PTMs, such as citrullination, carbamylation or glycosylation, on antigen processing/presentation becomes possible without the impact of the fluorophore. In this presentation, several applications of such methodology will be discussed. For instance, we, for the first time, used bioorthogonally labelled recombinant Vinculin, a Rheumatoid Arthritis auto-antigen, to study the effect of citrullination and carbamylation on antigen presentation in DCs. We found differences in antigen degradation rates in murine DCs in vitro. However, these did not translate into significantly altered T cell activation by human DCs.