CTLA-4 is an essential negative regulator of T cell activation and shares its costimulatory ligands with the stimulatory receptor CD28.  Blockade of CTLA-4 by monoclonal antibodies is an effective strategy for cancer immunotherapy nevertheless measuring CTLA-4 function in the in vitro setting can sometimes be challenging. In this poster we outline some mechanistic insights that have been obtained through an imaging approach. This reveals the dynamic intracellular trafficking of CTLA-4 and importantly its ability to capture and degrade costimulatory molecules from opposing cells. This has been used to design functional flow cytometric studies to reveal CTLA-4 activity and demonstrate the effects of antibody blockade. This combination of mechanistic insight and functional prediction is a powerful approach when designing assays to test costimulatory molecule function.