High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates

Abstract

T-cell activation via antigen presentation is associated with the formation of a macromolecular membrane assembly termed the immunological synapse (IS). The genesis of the IS and the onset of juxtacrine signalling is characterized by the formation of cell membrane microclusters and the organization of such into segregated microdomains. A central zone rich in T-cell receptor (TCR)-major histocompatibility complex microclusters termed the central supramolecular activation cluster (cSMAC) forms the bullseye of this structure, while the cellular interface surrounding the cSMAC is characterized by regions enriched in adhesion and co-stimulatory molecules. In vitro, the study of dynamic TCR microcluster coalescence and IS genesis in T-cell populations is hampered by cell migration within the culture system and resolution constraints resulting from lateral cell-cell contact. Here, we detail a novel system describing the fabrication of micropit arrays designed to sequester single T-cell-antigen presenting cell (APC) conjugates and promote IS formation in the horizontal imaging plane for high-resolution studies of microcluster dynamics. We subsequently use this system to describe the formation of the cSMAC in T-cell populations and to investigate the morphology of the interfacial APC membrane.

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 rearrangement of microclusters at the IS in a cell–cell system.

Figure 2.

The fabrication regime for micropit substrates (a) An array of circles is written in a 3 µm thick resist layer via photolithography. (b) The pattern is developed and etched via reactive ion etching (RIE), forming a master mould in silicon. (c) The master pillar mould is used to transfer an array of pits into a PDMS-coated cover-glass. (d) Experimental micropit substrates were 40 µm in depth and 20 µm in diameter. (e) aAPCs and T-cells were sequentially loaded into the pits forming cell conjugates for imaging. (Online version in colour.)

Figure 3.

Master substrates fabrication and transfer fidelity. (a) Micropillar master substrates possessed a uniform distribution of pillars arranged in a square conformation. Pillars were measured to be 40 µm high and possessed defined widths of 15–23 µm. (b) Imprinting of the pillar substrates resulted in the formation of patterned pit arrays as defined by the master substrate pillar dimensions. Micropit substrates were fabricated on microscopy cover-glasses laminated with a 50 µm deep spin-coated film of PDMS.

Figure 4.

Micropit loading with aAPCs. (a,b) K562 cells were introduced to the micropit substrates and allowed to settle by gravity for 30 min. Non-sequestered cells were subsequently removed by substrate washing. Bar = 100 µm. (c) The number of sequestered aAPCs was a function of pit diameter. PDMS pits with a diameter >20 µm introduced increased peri-pit area to the system, evident in 23 µm diameter pits which failed to retain sequestered cells following substrate washing. (d) Initial T-cell conjugation to aAPCs was associated with a flux in intracellular calcium levels. Mean fluorescence intensity was increased by approximately a factor of 3 in conjugated T-cells (line with filled circles) relative to non-conjugated cells (continuous line). Results are ±s.d. (Online version in colour.)

Figure 5.

Analysis of CD3–TCR microcluster recruitment during cSMAC genesis. (a) DIC and (b) the corresponding confocal laser-scanning confocal image of cellular conjugation in microfabricated pits. Vertical ‘stacking’ of aAPCs and T-cells facilitated microcluster analysis and cSMAC imaging within the horizontal imaging plane. Red outline, underlying K562 cell; green, Jurkat T-lymphocyte; orange, anti-CD3. Bar = 20 µm. (c) Both total (unfilled bars) and cSMAC-associated (filled bars) TCR cluster area dynamically increased at the immunological interface following cell conjugation. TCR microclusters became enriched at the immunological interface 15 min post conjugation, forming a central cSMAC at t = 30 min. Following 1 h, the mean diameter of the cSMAC had increased to approximately 20 µm². (d) TCR microclusters increased in frequency as post initial conjugation approached t = 15 min. Microclusters began to coalesce following 15 min to form larger, less numerous TCR aggregations. Results are ±s.d.

Figure 6.

The dynamics of TCR microcluster recruitment during cSMAC genesis. Representative time-lapse series of microcluster dynamics. TCR microclusters are actively recruited to the immunological interface before coalescing into a central cSMAC. The terminally formed cSMAC is marked by punctuate microdomains and regions of TCR void. Numerical values represent total minutes post initial cellular conjugation. Bar = 10 µm. (a,b) Microclusters become less diffuse and less frequent at the cell periphery as time increases. Microclusters form a central (dx = 0) high-signal cSMAC aggregation as measured by mean fluorescence intensity. (Online version in colour.)

Figure 7.

A pSMAC podosomal ring circumscribes the cSMAC by the aAPC. (a) Rendering a confocal Z-stack revealed the recruitment of actin to the IS. Green, actin; yellow, colocalization of TCR and actin. (b) SEM imaging of the cell–cell interface revealed this actin recruitment to be associated with the formation of a complex of podosome extensions at the IS. (c,d) Podosomes form a ring-like structure that circumscribes the IS at the pSMAC as identified by SEM and confocal imaging, respectively. Red, TCR; green, actin. Bar = 10 µm. (e) Pearson's coefficient was used to analyse the colocalization of actin to TCR microclusters. Pearson's coefficient was then plotted as the function of dx (pixel shift) to obtain a cross-correlation function, indicating minimal colocalization.