Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

This method can help to answer key questions in the human immunological field, such as how communication between leukocytes occurs on the molecular and cellular level. The main advantage of this technique is that unpurified human T cells can be analyzed ex vivo in a relatively short time period. Demonstrating this procedure will be Henning Kirchgessner, a technician from our laboratory.

Begin by mixing one milliliter of freshly drawn human peripheral blood with 30 milliliters of ACK lysis buffer in a 50-milliliter conical tube. After eight minutes at room temperature, fill the tube with PBS for a centrifugation wash and resuspend the pellet in two milliliters of culture medium for a 60-minute incubation at 37 degrees Celsius. At the end of incubation, add five times 10 to the fifth Staphylococcus aureus enterotoxin B or SEB-loaded or unloaded Raji cells in 500 microliters of culture medium into individual FACS tubes, followed by 650 microliters of pan-leukocytes.

Spin down the co-cultures, and resuspend the pellets in 150 microliters of fresh culture medium for a 45-minute incubation at 37 degrees Celsius. Then, gently vortex the cells for 10 seconds at 100 rpm while adding 1.5 milliliters of 1.5%paraformaldehyde. After 10 minutes at room temperature, stop the fixation with one milliliter of PBS supplemented with 1%BSA and collect the cells by centrifugation.

Resuspend the pellets in 100 microliters of PBS plus BSA and 0.1%saponin, and add the cell samples to two individual wells of a 96-well, U-bottom plate. After 15 minutes at room temperature, centrifuge the plate and resuspend the pellets in 50 microliters of PBS plus BSA and saponin containing the fluorophore-conjugated antibodies or compounds of interest for a 30-minute incubation in the dark at room temperature. At the end of the incubation, wash the cells three times in 150 microliters of PBS plus BSA and saponin and resuspend the pellets in 60 microliters of PBS.

To image the cells by flow cytometry, open the analysis software, check the liquid level, and click Startup in the Instrument menu. Next, click Load, and load the samples into the port when prompted. Under the Illumination tab, change the excitation laser power to the appropriate nanometer lengths.

Then, adjust the gates for channels four and 11, and adjust the area for channel one. To evaluate the gating and laser power adjustments, switch the View menu to the respective gate and adjust the gates and laser powers until the cells and cell couples are visible. Then, in the File Acquisition tab, define the sample name and the number of images to acquire and the appropriate gate for CD3 staining and click Acquire to begin the acquisition.

To analyze the acquired cell images, open the appropriate analysis software. Following the instructions in the Compensation dropdown, produce a compensation matrix and save the matrix as comp date ctm. Next, open a sample raw image file, and apply the comp date ctm in the window to generate the compensated image and default data analysis files.

After opening the compensated image files, convert the images to color mode. Adjust the lookup tables to obtain optimal visible colors in the Image Gallery Properties toolbar. Open the Mask Manager in the Analysis menu, and define the T cells by selecting a 60%Threshold mask for channel five and filling the mask to create the T cell mask.

Next, select Valley mask for channel two with a width of three pixels to create the Valley mask, and combine the T cell and Valley masks to create the T cell synapse mask. To calculate the total CD3 expression in T cells, open the Feature Manager and create the feature Intensity, T cell, channel five. To calculate the total amount of F-actin in the T cells, create the feature Intensity, T cells, channel six.

To evaluate the CD3 amount in immune synapses, create the feature Intensity, T cell synapse, channel five. To determine the amount of F-actin in the immune synapse, create the feature Intensity, T cell synapse, channel six. Finally, to define the F-actin and CD3 enrichment, calculate the ratios of F-actin or CD3 in the immune synapse and the total expression of the selected protein, respectively.

In these graphs, an overview of the critical gating strategy for quantifying the protein enrichment in the immune synapse between the surrogate antigen-presenting cells, or APCs, and the T cells in unpurified pan-leukocytes taken from a low-volume human blood sample is shown. Here, representative images of T-cell-APC conjugates with low levels of CD3 and F-actin within their immune synapses in the absence of SEB are shown, whereas these T-cell-APC conjugates demonstrate a strong CD3 and F-actin enrichment in the presence of SEB. In the absence of superantigen, 15%of the T cells exhibited enrichment of both CD3 and F-actin at the immune synapse, whereas a 29%enrichment is observed in the presence of superantigen.

Indeed, in the absence of superantigen, less than 20%of the total F-actin in the cells accumulates at the immune synapse, with over 30%observed at the synapse in the presence of superantigen. Notably, CD3 accumulates at the immune synapse even in the absence of superantigen, although this accumulation is also significantly increased by the addition of superantigen, demonstrating the fitness of this method for quantifying protein accumulation at the T-cell-APC immune synapse. Once mastered, this technique can be completed in six to seven hours if performed properly.

While attempting this procedure, it's important to remember to include all relevant controls, including samples without superantigen, as protein enrichment also occurs if APCs without SEB are used. Following this procedure, other methods like super-resolution microscopy can be performed to answer additional questions about the fine structure of the immune synapse. After its development, this technique paved the way for researchers in the field of cellular communications to explore the immune synapse in humans for basic research, clinical trials, and translational science.

After watching this video, you should have a good understanding of how to analyze the T-cell-APC connective zone and the immune synapse in human T cells ex vivo. Don't forget that working with primary human materials can be hazardous and that precautions such as wearing gloves or working under biosafety level two conditions should always be taken while performing this procedure.