An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

The overall goal of this technique is to utilize endothelial cells as a novel type of planar antigen presenting cell model to investigate fundamental antigenic signaling processes. This method can help us address key questions in the field of adaptive immunity, such as how subcellular and molecular remodeling dynamics are coordinated spatially and temporally within the immunological synapse. The main advantage of this technique is that the endothelium forms of virtually planar a PC surface.

This allows for imaging with optimal spatiotemporal resolution within the context of a physiologically complex and a formable cell surface. Though this method focuses on CD four positive TH one effector memory cell activation, it can also be applied for the study of a variety of T-cell types antigen and signaling processes. Today, Chris and I will show you to perform the second Begin by code transecting primary human lung or dermal microvascular endothelial cells with membrane YFP and soluble DS red via NU nuclear affection and plate the transfected cells onto live cell imaging culture plates.

The next day replace the cell culture medium with fresh medium supplemented with interferon gamma to induce M MHC two expression on the endothelial cells, followed by stimulation of the cells with 20 nanograms per milliliter of TNF alpha 24 hours later on day three 30 to 60 minutes. Before starting the experiment, incubate the endothelium with one microgram per milliliter, each of the bacterial superantigens staphylococcal enterotoxin B and toxic shock syndrome toxin one at 37 degrees Celsius. During the incubation, count the number of viable cells from the lymphocyte culture of interest and transfer two times 10 to the sixth of the cells into a 15 milliliter conical tube.

Next, spin down the cells and resuspend the pellet in two milliliters of freshly prepared buffer A such that no cell clusters remain. Then add two micromolar RA two to the T-cell sample, cap the tube and immediately invert it to homogeneously. Distribute the dye throughout the cell suspension.

After a 30 minute incubation at 37 degrees Celsius, spin down the labeled lymphocytes and gently but thoroughly resuspend the cells in 20 to 40 microliters of fresh buffer A to live image, the cells turn on the microscope system and open the appropriate image capture software. Set the appropriate parameters for automated multi-channel time-lapse imaging, as well as the interval for acquisition for 10 to 30 seconds and a total duration of approximately 20 to 60 minutes. Next, add fresh oil to the objective.

Mount a microscope dish containing 0.5 milliliters of buffer A onto the heating stage adapter, and immediately switch on the adapter previously set to 37 degrees Celsius. After two to three minutes of equilibration, use a 20 microliter pipette to add the resting RA two loaded lymphocytes into the mounted microscope dish chamber and turn on the bright field imaging. Select the light path to the eye pieces and use the course focus knob to bring the objective into contact with the bottom of the microscope dish.

Then use the eyepiece and the fine focus knob to locate the T cells settled at the bottom of the dish and use the XY stage controls to select a field containing at least 10 cells when the appropriate cells have been located. Switch from brightfield to the fluorescent light source and from IP imaging to the CCD camera. Optimize the acquisition parameters and then acquire resting Fira 2 3 40 and FIRA 2 3 80 images.

One of the key steps to generate successful results is careful optimization of the fluorescence acquisition parameters, ensuring that sufficient signal is collected and in the case of fira two, that the baseline 3 43 80 ratio is close to one. When the FIRA two exposure times have been set, replace the dish of T cells with a microscope dish of endothelial cells using a disposable transfer pipette rapidly remove the media and rinse the cells once with one milliliter of prewarm buffer A, then replace the wash with 0.5 milliliters of fresh buffer A.Using the objective identify fields in which the brightly fluorescent endothelial cells appear healthy. Then adjust the acquisition parameters for membrane YFP and DS RED as just demonstrated for fira two.

Taking care that the mean fluorescent signal intensity in each channel falls between 25 and 75%of the dynamic range of the detector. Before conducting the live cell imaging experiment, capture several intervals of images of the endothelial cells alone to establish a baseline with the automated software. Then inserting the tip of a small volume into the media close to the center of the objective.

Slowly dispense five microliters of concentrated fira, two loaded lymphocytes to the center of the microscope dish imaging field. As the lymphocytes settle, make fine adjustments to the focus to ensure that the T-cell endothelial cell interfaces are maintained in the focal plane. With the 40 and 63 x objectives, 10 to 20 cells per field is optimal.

After the desired observation interval, continue imaging and immediately pipette CIN directly into the microscope dish to a final concentration of two micromolar to induce a maximal calcium flux sphera. Two signal. When applying treatments to cells, take care to maintain sharp focus on the apical membrane YFP labeled surface of the endothelium to ensure optimal resolution of the immune synapse remodeling.

In the absence of superantigen on the endothelial superantigen specific CD four positive TH one lymphocytes rapidly spread polarize and laterally migrate over the endothelial cells. During this process, the lymphocytes extend short-lived protrusions or ILPs into the endothelial surface, which are evident as rings of membrane YFP fluorescence analogous to footprints in the presence of superantigen, close cell to cell contacts driven by the ILPs are associated with the initiation of a calcium flux leading to an accumulation of stabilized ILPs within the immunological synapse. Such ILP arrays are coupled to sustained calcium signaling.

The rings of membrane fluorescence that form during T-cell endothelial cell interactions correspond to the T-cell protrusions driving discrete invagination spots in endothelial cells as confirmed by the demonstration that these rings correlate spatially and kinetically with the zones of displaced and excluded cytoplasm. Within minutes of co incubation of the endothelial and T-cells in the presence of superantigen adhesion and signaling molecules associated with antigenic signaling are coen enriched within the pota prints and ILPs of the immunological synapse. Indeed, the T-cell endothelial immunological synapse exhibits a discreet three-dimensional architecture punctuated by T-cell ILPs extending into the antigen presenting cell surface within which the antigen recognition molecular machinery is enriched.

With this approach, key obstacles within existing a PC models can be overcome, allowing observation of otherwise undetectable 3D subcellular architecture and the rapid remodeling of physiologic T-cell a PC interfaces. This offer potential for new levels of understanding of antigenic signaling processes.