Imaging the immune synapse: three-dimensional analysis of the immune synapse

Abstract

T cell receptor (TCR) stimulation of T lymphocytes by antigen bound to the major histocompatibility complex (MHC) of an antigen-presenting cell (APC), together with the interaction of accessory molecules, induces the formation of the immunological synapse (IS), the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. Upon IS formation, an initial increase in cortical filamentous actin (F-actin) at the IS takes place, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. These reversible, cortical actin cytoskeleton reorganization processes that characterize a mature IS occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. Besides, IS formation constitutes the basis of a signalling platform that integrates signals and coordinates molecular interactions that are necessary for an appropriate antigen-specific immune response. In this chapter we deal with the three-dimensional (3D) analysis of the synaptic interface architecture, as well as the analysis of the localization of different markers at the IS.

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse

“…S10). Profile analyses of MFI corresponding to each separate channel and the colocalization pixels at the IS interface (ZX axes) were performed by using "Intensity Profile" tool in NIS-AR and then Excel, as reported (84).…”

“…Video 5 and Fig. 7), generated as described (84). The areas of the F-actinlow region at cIS (Fact-low cIS area) (yellow line) and the synapse (IS area) (white line) (i.e., Fig.…”

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse

“…2D colocalization analyses were accomplished by using JACoP plugin from ImageJ, whereas 3D colocalization and the corresponding videos to generate the IS interface were performed using the "colocalization" tool, followed by the "volume view" and then "movie maker" tools from NIS-AR. For 3D colocalization analyses, the original emission colors of the different fluorochromes were changed to Red, Green or Blue, since NIS-AR requires these colors for 3D colocalization (47). In particular, F-actin and Phospho-Ser PKC substrate acquired in magenta were changed to 34 blue, and YFP acquired in yellow was changed to green (i.e.…”

“…Profile analyses of MFI corresponding to each separate channel and the colocalization pixels at the IS interface (ZX axes) were performed by using "Intensity Profile" tool in NIS-AR and then Excel, as reported (47). To measure the relative size of the F-actin low area at the cIS, which quantifies the decrease in F-actin density at the cIS, we used the last frame from the former IS interface videos (Suppl.…”

“…Video 2 and Fig. 7), generated as described (47). The areas of the F-actin-low region at cIS (Fact-low cIS area) (yellow line) and the synapse (IS area) (white line) (i.e., Fig.…”