Imaging the Human Immunological Synapse

Abstract

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90° angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

Inducible Polarized Secretion of Exosomes in T and B Lymphocytes

“…Stimulation with a heterologous receptor agonist, that mimics TCR-derived signals leading to full T cell activation [78] and AICD [79], also induced exosome secretion in CD4 + Jurkat cells [67], suggesting that exosome secretion is a general consequence of T lymphocyte activation. IS formation by CD4 + Jurkat cells and superantigen-coated Raji B cells acting as an APC, which constitutes a well-established IS model [61,80,81], induces polarized MVB traffic towards the IS, MVB degranulation and exosome release [15,18] (Figure 2, right side panel). In this Th-APC IS model, a positive role of TCR-triggered DAG and its regulator DGKα [70], in polarized MVB traffic towards the IS was demonstrated [15,67,82].…”

Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity

“…Total internal fluorescence (TIRF) microscopy has limited z-axis resolution but is quite useful for analyzing the IS, which occurs in a narrow plane ( 145 , 146 ). Two-photon fluorescence microscopy (TPFM) can complement TIRF by allowing similar resolution but with the ability to look at subcellular properties ( 117 , 147 ).…”

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

“…We have previously shown, by using a well-established IS model [31] [35] [32], that PKCδ-interfered Jurkat clones challenged with antigen on APC secreted a lower amount of exosomes upon IS formation [14]. We also defined that the deficient polarization of MVB containing the exosome precursors towards the IS underlies the decreased exosome secretion [14].…”

“…Jurkat clones transfected with the different expression plasmids were attached to ibidi microwell culture dishes using fibronectin (0.1 mg/ml) at 24-48 h post-transfection, and stimulated in culture medium at 37 ºC. In some experiments requiring IS formation, Raji cells attached to ibidi microwell culture dishes using fibronectin (ibiTreat, for paraformaldehyde fixing) or poly-L-lysine (glass bottom, for acetonefixation) were labelled with CMAC and pulsed with 1 µg/ml SEE, mixed with transfected Jurkat clones and immune synapses analyzed as described [31] [15] [32].…”

“…[32].For quantification, digital images were analyzed using NIS-AR (Nikon) or ImageJ softwares (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2004). The quantification and analysis of F-actin mean fluorescence intensity (MFI) in a centrosome-centered area (centrosomal area F-actin MFI) in time-lapse experiments, was performed within a 2 µm diameter, floating region of interest (ROI) (i.e., ROI changing XY position over time), centered at the center of mass of the MTOC (MTOC c ), by using NIS-AR software (Fig.…”