The overall goal of this procedure is to measure T cell alloreactivity using imaging flow cytometry. This method can help answer key questions in transplant immunology, such as what proportion of a recipient's T cells are reactive to donor alloantigens. This technique combines the multiparametric quanititative powers of flow cytometry with the imaging capabilities of fluorescent microscopy to allow the high-throughput examination of individual T cell antigen presenting cell synapses.
Demonstrating the procedure will be Sajad Moshkelgosha, a postdoc from my laboratory. Begin by seeding zero point five times 10 to the sixth dendritic cells. into each well of a 96-well cell culture plate, followed by one times 10 to the sixth T cells, and a final volume of up to 50 microliters total of culture medium per well.
Incubate the cold cultures for four hours at 37 degrees Celsius and five percent carbon dioxide. Then, add three times the culture volume of one point five percent formaldehyde in PBS to each well for 30 minutes at room temperature. At the end of the fixation, transfer the cells from each well into a corresponding five milliliter polystyrene tube.
Stain the cells with the appropriate fluorochrome conjugated antibodies in 100 microliters of wash buffer for 30 minutes at room temperature protected from light. Next, wash the cells in one milliliter of wash buffer and resuspend the pellets in perm/wash buffer containing phalloidin FITC. After another 30 minutes at room temperature protected from light, wash the cells in one milliliter of fresh perm/wash buffer.
Resuspend the pellets in the nuclear dye of interest in perm/wash buffer for a final 30 minute incubation at room temperature protected from light. Wash the cells in one millimeter of perm/wash buffer followed by one wash in wash buffer alone. Then, resuspend the cells in 50 to 100 microliters of fresh wash buffer.
Transfer the cells into small capped microcentrifuge tubes. To analyze the cell-to-cell interactions, first reserve one channel for brightfield image acquisition and load the first control tube. With the brightfield channel turned off, in the workspace window, create a new scatterplot for each channel used with the aspect ratio over the area.
Ingate the singlets where the aspect ratio is close to one. For each fluorochrome, check the positive population and adjust the laser voltage in the illumination box, as necessary. In the channels box, select the appropriate channels for the fluorochromes used in the staining.
Then, click record. When the number of events reaches the specified threshold, the acquisition will stop automatically. After all of the single stain controls have been acquired, load the first experimental sample tube and acquire several tens of thousands of events under the appropriate analysis channels, including the brightfield channel.
To generate a compensation matrix, load the single stain control data files into the compensation wizard and select the appropriate fluorescent channels for the experiment. After confirming that the correct positive populations have been selected, double-click a value in the matrix and add the graph to the analysis area for each channel. Click finish to save the compensation matrix as a CTM file.
Load the RIF file into the appropriate analysis software. Convert the RIF file to a data analysis DAF file. Open the first sample file.
Plot the root mean square of the rate of change of the imaging intensity in the brightfield channel. Then, plot the aspect ratio versus the area for the antigen-presenting cell marker. Ingate the doublets containing a single antigen-presenting cell.
Plot the aspect ratio versus the area for the T cell marker. Ingate on the doublets containing a single T cell. To identify the cells in contact with each other, under the analysis menu, use the masks option to open the masks manager, and name the new mask T cell object mask.
Click the function button and in the dialog box, select object. Select the channel in which the T cell marker is detected and click OK.Create a mask for the antigen-presenting cell marker in the appropriate channel in the same manner. Plot the T cell marker fluorescence intensity in the antigen-presenting cell object mask against the antigen-presenting cell fluorescence intensity in the T cell object mask.
Then, draw a gate that includes only the cells in contact with one another. Finally, manually review the phalloidin FITC images of the events within this gate to distinguish the mature immune synapses from the simple cell-to-cell contacts using the tag images function to mark these images. Here, the membrane contact gate is shown for splenic CD4 positive T cells obtained from tolerized and nontolerized recipients of B6 cardiac allografts seven days post transplant and coincubated with B6 bone marrow derived dendritic cells, as demonstrated.
With the synaptic and nonsynaptic events indicated by the green crosshairs. Brighfield and fluorescent channel analyses also allow visualization of the individual synaptic and nonsynaptic events. Indeed, synapses from both non-tolerized and tolerized CDA recipients of B6 hearts are easily distinguishsed from nonsynaptic contacts by the presence of dense FITC positive ridges at the T cell antigen-presenting cell interface.
After further development, this technique may be able to be applied in clinical settings, such as, in the reduction or withdrawal of immunosuppresssion or the prediction of graft outcome in human translpant recipients.
