With optical trapping of cells demonstrated, the next important step was to demon- strate manipulation of biological events during fluorescence imaging. Here, we turned to the induction of immunological synapses, an important immune event within the body that mediates immune function.
7.3.1
The immunological synapse
Regulation of the body’s immune response is through cell-cell interactions. Antigen- presenting cells (APCs) capture antigens from their environment, process them, and present them on their membranes in a readable format for T cells. T cells will junction to an APC and act depending on the familiarity of the presented antigens. If a pathogen is detected then the T cell can turn cytotoxic and kill the cell or secrete cytokines to recruit pathogen-killing cells to the area [255].
The junction between a T cell and an APC is known as an immunological synapse (IS). A synapse is initiated when a T cell receptor (TCR) is stimulated by an epitope (fraction of the captured antigen) and major histocompatibility complex molecule pair on an APC membrane in the presence of a co-factor [255]. During a synapse, the T cell will polarise. Actin within the cell remodels to form a ring at the periphery of the synapse to stabilise the synapse. The microtubule organisation centre (MTOC) remodels close to the IS, which brings the Golgi and cytotoxic granules (in the case of Natural Killer cells) proximal to the IS to allow for polarised secretions of cytokines or granules [256]. ISs also mediate exchanges of proteins, nucleic acids [257] and vesicles (which also polarise to the IS [258]) and are thought to regulate gene expression within the immune system, making the IS much more multifunctional than previously thought.
A typical IS experiment uses a population of synapsing cells; by scanning the field of view, it is possible to infer temporal information from the proportion of synapses at each stage. Not only is this process laborious, it also has poor temporal resolution. The manipulation of individual cells to induce ISs allows us to follow the synapse from induction to termination. Micro-manipulators have been used for IS induction and subsequent force measurement [259] but this is slow (only 3-5 cells were used for each experiment) and requires experienced users. Ease-of-use and the potential for automation and computer control makes optical tweezers an attractive method for IS induction. Indeed, optical tweezers have been used for a variety of cell manipulation experiments [81, 82, 260].
SR imaging of actin and lytic granules at Natural Killer cell ISs has been well investigated using fixed cells [261, 262] with both STED and SIM. The majority of these studies involved imaging fixed cells synapsing with an antigen-coated substrate or lipid bilayer, while this brings the IS close to the imaging interface for better optical imaging, it does not adequately represent a synapse in 3D space in an in vivo setting.
The mechanisms behind the MTOC and endosomal remodelling to the IS is poorly understood, developing a protocol to induce ISs at will and perform live-cell imag- ing, using an integrated optical trap and SR microscopy, would greatly advance our knowledge of this important process.
7.3.2
Immunosynapse induction by optical trapping
One of the markers for the start of an immunological synapse is an increase in calcium in the T cell. This is an almost instantaneous effect, which makes it a useful marker for the onset of an IS, remodelling is a more subtle and slower effect (at least ten minutes), which might be difficult to observe during an experiment. A calcium fluorophore, Fluo-4 (Life Technologies, USA), which increases in fluorescence when it binds to calcium, was used to detect changes in cytosolic calcium. An optical trap was employed to induce
a single bead-cell IS and the calcium response was monitored. 3 µm polymer beads (Spherotech, USA) were conjugated with mouse antibodies (eBioscience, USA): anti- CD3 bypasses the TCR and directly activates the B3Zs, and anti-CD28 is a co-factor to ensure sustained synapse induction (unlike in Wei et al. [234] and Tam et al. [81] where beads coated in anti-CD3 only are used) [255, 263].
The antibody conjugated beads adhered to the coated-glass surface. It was therefore easier to move the cell towards the bead than the bead towards the cell. The imaging and trapping planes were co-aligned so the cells were in focus when trapped, improving imaging of the cells when trapped. Epi-fluorescence images using the FITC filter were taken every 50 ms for 160 s using a 20x objective. Region of interest intensities were measured at each time point using ImageJ and background corrected. Photobleaching correction was performed by fitting an exponential curve by least squares optimisation to the normalised control cell signal and dividing the normalised activated response by the fitted curve.
An optical trap-induced immunosynapse and subsequent calcium increase is shown in Figure 7.9. (A) shows a bright cell (B3Z) with a polymer bead attached to it to the left of the field of view and a dark cell (right) that is yet to be activated (low fluorescence), with a bead present not currently visible. The dark cell was brought towards a bead adhered to the bottom of the dish using the optical trap (B). The bead becomes visible as the cells begins to increase in fluorescence intensity (C). Between 10 and 160 seconds, the fluorescence increases six-fold, signalling the creation of an immunosynapse as the T cell is activated (D). No increase in calcium levels were seen when trapping cells in the absence of beads.
While it was possible to demonstrate cell trapping and the induction of an im- munosynapse with the trapping system described above, the next step of incorporating super-resolution imaging to formation of an IS was not achieved during this study. The adhesion of cells and beads to the dish made trapping and moving difficult. Coating the dishes made cells tend to move away from each other even when placed close together,
0.1 0.3 0.5 0.7 0.9 1.1 1.3 0 50 100 150 I ( ar b . u n it s) Time (s) Control PB fit Activated A B C D
Figure 7.9 – Use of an optical trap to induce an IS by moving a Fluo-4 loaded B3Z towards an anti-CD3 anti-CD28 coated bead. (A) Unactivated T cell (right) prior to synapse formation with an antibody coated bead. (B) Slight increase in fluorescence as an IS is initiated. (C) Larger increase as the synapse matures. (D) Increase in fluorescence of the activated cell (red), where every point plotted represents the average of ten measured points. The activated cell’s fluorescence was corrected for photobleaching by dividing by the PB line (dashed) fit to the control cell (blue).
as performed in McNerney et al. [82]. Adherent target test cells (HeLa) displayed in- visible protrusions, preventing close contact with the trapped suspension cell. When adding antibody-coated beads to a dish of cells, no tubulin, mitochondrial or endosomal remodelling was observed. The stains [264] and beads [265] used should be sufficient to induce remodelling so the reason behind this behaviour is currently unknown. More experiments with other cell types and different incubation protocols would perhaps extricate the reasons behind this failure to polarise.