Juxtacrine signalling and cell–cell interactions involve multiple adhesion and regulatory molecules, the collective interaction of which can direct cell activation and differential cell function. In particular, T-cell recognition and activation is mediated by clonotypically distributed αβ and γδ T-cell receptor (TCR) molecules that interact with peptide-loaded major histocompatibility complexes (MHCs) presented on the antigen-presenting cell (APC) membrane [1
]. The antigen-specific chains of the TCR do not possess signalling domains per se
but instead are coupled to the multi-subunit signalling apparatus CD3, to form the TCR–CD3 receptor complex [2
]. The mechanism by which TCR ligation can directly regulate the T-cell signalling apparatus remains elusive in immunology. It seems clear however that a sustained T-cell response involves the engagement of multiple co-stimulatory and adhesion membrane receptors, TCR oligomerization and a high-order arrangement of TCR–MHC complexes at the T-cell–APC interface.
Coined by C. Sherrington from the Greek ‘syn’ (together) and ‘haptein’ (to clasp) to signify neuronal cell–cell junctions [4
], the term immunological synapse (IS) was first extended to T-cell biology by M. Norcross to describe the interfacial interaction that occurred between a T-cell and an antigen-presenting B-cell [6
]. Later, Kupfer and colleagues revealed a compartmentalization of the interactions at the interface of the T-cell and antigen-presenting membranes [8
], whereby signalling and adhesion molecules self-organize into concentric regions at the IS. A central TCR–MHC-rich zone termed the central supramolecular activation cluster (cSMAC) forms the bullseye of this structure, while the cellular interface surrounding the cSMAC, termed the peripheral supramolecular activation cluster (pSMAC), denotes an outer region enriched in cell–cell adhesion molecules. Parallel studies with supported planar bilayers have led to the proposal that the active organization of segregated adhesion molecules and antigen receptors constitute the hypothesized IS [9
], and that this segmented organization is important in the regulation of lymphoid juxtacrine signalling processes. Studies to date suggest that the macrostructure of the cSMAC is formed from the centripetal streaming of plasma membrane microclusters, TCR aggregates which measure approximately 1 µm in diameter [11
]. Further to this, evidence suggests that the spatial organization of the clustering within the IS plays an active role in regulating the signalling state of individual molecular components, and thus can alter T-cell activation [14
In vitro study of the genesis of the IS and the dynamic process of TCR microcluster coalescence in T-cell populations is hampered by cell migration within the culture system as well as by resolution constraints resulting from lateral cell–cell contact relative to the normal trajectory of the incident light (a,b). At present, high-resolution imaging of the IS in a live cell system relies on the use of several stratagem to facilitate the study of TCR microcluster dynamics, all of which are associated with specific advantages and drawbacks.
Figure 1. Imaging the immunological synapse. (a,b) Lateral orientation of the T-cell–APC interface prevents detailed analysis of microcluster dynamics. Green, Jurkat T-lymphocytes; blue, immune synapse (TCR). (c) Ideal plane of imaging to study the dynamic (more ...)
Confocal volume rendering is commonly used in biological confocal imaging and uses a combination of optical and computational techniques to acquire a series of x
-plane images, which are recorded along the z
-axis and the signal volume rendered for quantitative analysis. Computer algorithms are used to compensate the absence of interplane detail and to reconstruct a three-dimensional image from a rendered z
]. This method is commonly employed to study the dynamics of IS formation in lateral T-cell–APC conjugates [8
], and can be used to rapidly acquire three-dimensional information of the IS morphology and structure. Deconvoluted and three-dimensional rendered images however suffer from artefactual distortion, and a loss of resolution in the z
-dimension. As a result, reliable interpretation of these images may be difficult or even impossible, particularly in dynamic live-cell systems [20
Laser tweezer or laser trap technology has recently been implemented for the manipulation of T-cell–APC conjugates in order to resolve dynamic microcluster localization to the IS [21
]. Importantly, this system allows vertical cell orientation, placing the IS within the horizontal x
-plane for high-resolution imaging experiments [22
]. This system suffers however from extremely low throughput owing to the manual intricacies needed for cell capture and alignment, which is carried out in single cell conjugates. A further limitation of this method arises from cellular phototoxicity complications, resulting from prolonged cellular exposure to the trap beam or thermotoxicity owing to local heating of the medium.
As mentioned above, supported lipid bilayers formed on glass surfaces have driven current models for IS formation and sustained T-cell signalling [23
]. The continuous fluid lipid bilayer forms from the spontaneous self-assembly of liposomes or proteoliposomes on a clean glass surface, and imitates the phospholipid bilayer of the APC membrane [24
]. Membrane proteins can subsequently be inserted into the bilayer and undergo the free lateral mobility that is inherent to native cell membrane proteins [25
]. Additionally, the well-defined planar interface facilitates high-resolution imaging, particularly by total internal reflection microscopy [28
]. While the bilayers provide a powerful approach, testing the predictions from this model system requires high-resolution imaging of the T cell–APC interface. Further, other phenomena requiring membrane dynamics of the APC are lost when the APC is replaced by a supported lipid bilayer.
Recently, Maus et al
] have described an artificial APC (aAPC) system derived from the chronic myelogenous leukaemia line K562 and subsequently transduced to express an array of T-cell stimulatory ligands. K562 cells do not naturally express MHC or T-cell co-stimulatory ligands, yet do retain many other attributes that make dendritic cells (DCs) such effective APCs, such as cytokine production, expression of the adhesion molecules ICAM-1 and LFA-3 that enhance T-cell–APC interactions, and macropinocytosis. These cells can be readily transduced with a library of lentiviral vectors for the customized expression of stimulatory and co-stimulatory molecules that can be used to activate and expand different subsets of T cells.
Here we detail a novel system describing the fabrication of micropit arrays designed to sequester a single T-cell–aAPC conjugate and promote IS formation in the horizontal imaging plane for the high-resolution study of TCR microcluster dynamics (c
). We used K562-based aAPCs which expressed multiple gene inserts, including human lymphocyte antigen (HLA)-A2, CD64 (the high-affinity Fc gamma type 1 receptor) CD80, CD83, CD86, CD137L (4-1BBL) and CD252 (Ox40L), and have proved to be more efficient in activating and expanding CD8+ and CD4+ T-cells than the magnetic bead-based aAPC system [31
]. APCs were loaded with a fluorescently tagged anti-CD3 marker and T-lymphocytes were subsequently loaded onto the sequestered aAPC cells to study the dynamics of microcluster genesis and the recruitment of the TCR to the IS. The system successfully isolated and allowed large-scale analysis of the immunological interface in thousands of T-cell–APC conjugates, facilitating both fixed and live-cell imaging.