T cell activation is initiated when T cells recognize their cognate antigen on the surface of antigen presenting cells (APCs). This triggers formation of a specialized membrane interface termed the immunological synapse (IS) which governs the spatial organization of the intercellular protein interactions ultimately determining the T cell response. While this is a fundamental process in adaptive immunity, tools for quantitative analysis and visualization of the IS molecular architecture are lacking. Here we present isMap, a computational framework for automated cell segmentation and quantification of various parameters related to T cell activation and IS formation on supported lipid bilayers (SLBs), including fluorescence intensity measurements, colocalization analysis and radial averaging. We validate isMap by confirming previous results showing that CD58 initially clusters with T cell receptor (TCR) before segregating into a distal ring during synapse maturation in activated CD8 + T cells. We also show that PD-L1 is initially distributed across the IS before ultimately accumulating with TCR in the center of the fully mature synapse. ICOSL, CD80 and CD86 cluster in the center of the contact area through all stages of IS maturation and colocalize with TCR in the order of ICOSLCD86CD80. These findings demonstrate isMap’s utility in dissecting the functional organization of the IS and highlight the dynamic redistribution of ligand-receptor pairs during T cell activation.
Singleton et al. (Wed,) studied this question.