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.

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

“…To prevent biases, please try to randomly select as many synaptic conjugates from different fields as possible. Unfortunately, it is not easy to distinguish canonic IS from irrelevant cell contacts or cell aggregates randomly produced by the high cell concentrations used to favor IS formation (Bello-Gamboa et al, 2019). Evaluation of the cup-shaped profile of the effector T lymphocyte and/or lamellipodium formation observed in the transmittance channel may help to identify IS (Fernández-Hermira et al, 2023).…”

“…1 and 2). Please refer to the following references for further details, since this IS model has been exhaustively described (Montoya et al, 2002) (Alonso et al, 2011) (Bello-Gamboa et al, 2019). Endpoint fixation is performed with 4% paraformaldehyde at room temperature (RT) for 15 min, and subsequently with chilled acetone for 5 min (see Note 1 ).…”

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

“…With respect to FMNL1β translocation, the early reports (29) did not demonstrate IS-induced relocalization of FMNL1β, most probably because establishment of a mature, fully productive IS, is intrinsically a stochastic, rapid and asynchronous process difficult to image at its initial stages (46). Moreover, most early reports used an end-point approach that forces cell conjugate formation by centrifugation (52), and this approach is unable to analyse incipient IS (52) (46) (43). We have circumvented this caveat by using living cell imaging of spontaneously formed, emerging synapses at high-temporal resolution (46).…”

“…This is probably due to the fact that most early studies have used an end-point approach that does not allow to analyse the incipient IS (29) (52) (46) (43). Since FMNL1β is responsible for MTOC/MVB polarization to the IS (43), we analysed FMNL1β subcellular location in developing synapses by time-lapse, epifluorescence microscopy (46) (43). For this analysis, FMNL1-interfered, YFP-FMNL1βWT-expressing C3 Jurkat clone was challenged with SEE-pulsed Raji cells.…”

“…Time-lapse acquisition during the indicated times and analysis were performed using NIS-AR software (Nikon). Subsequently, in some experiments epifluorescence images were improved by Huygens Deconvolution Software from Scientific Volume Image, using the "widefield" optical option as previously described ( 46) (43). For quantification, digital images were analysed using NIS-AR (Nikon) or ImageJ software.…”