The overall goal of this protocol is to describe the use of photoactivatable peptide MHC to induce polarized signaling in T lymphocytes. This method can help answer key questions in immune cell signal transduction such as how lymphocytes transduce rapid localized signals and then mount polarized cellular responses to those signals. The main advantage of this technique is that it offers spatial temporal control of T cell signaling.
It does this by fixing the precise time and location of T cell receptor activation. Demonstrating the procedure will be Elisa Sanchez, a grad student from my laboratory. Begin by adding biotinylated poly-L-lysine, or bio-PLL, diluted one to 500 in distilled deionized water to eight-well chambered cover glass.
Incubate for 30 minutes at room temperature. Following the incubation, wash with distilled deionized water and then dry for two hours at room temperature. Block bio-PLL coated surfaces with blocking buffer consisting of HEPES buffered saline with 2%BSA for 30 minutes at room temperature.
Following the incubation, remove the blocking buffer from the bio-PLL coated surfaces without allowing the wells to dry. Then add streptavidin and incubate at four degrees Celsius. After an hour, wash the coated surfaces in HBS.
Fill and invert the chamber slide wells four to five times, removing HBS from the wells. Do not allow the wells to dry. Add the specific biotinylated photoactivatable peptide major histocompatibility complex ligands and adhesion molecules for either CD4 positive T cells or CD8 positive cells to the bio-PLL coated surfaces.
Protect from light and incubate for one hour at room temperature. After the incubation, wash as before and leave in HBS until ready to seed. Finally, add 200, 000 CD4 positive or CD8 positive T cells expressing the appropriate TCR into the correct wells and allow the cells to adhere at 37 degrees Celsius for 15 minutes.
Once the cells have attached to and spread on the surface, they are ready for photoactivation and imaging. Use an inverted total internal reflection fluorescence microscope outfitted with a UV-compatible 150X objective lens for image acquisition. Monitor probes in both the green and red channels using 488 nanometer and 561 nanometer excitation lasers, respectively.
After mounting the chamber slide containing the T cells, adjust the microscope settings to obtain TIRF or epifluorescence illumination, as necessary. In Live mode, select a field of cells that are expressing the fluorescent probes of interest. Establish micron scale regions for photoactivation beneath individual cells using software control.
Then, start the acquisition. Typically, 80 time points are required with an interval of five seconds between each. This leaves time for sequential 488 nanometer and 563 nanometer exposures in the case of dual color experiments.
Then, after acquiring 10 time points, photoactivate the selected regions by opening the digital diaphragm shutter for one to 1.5 seconds. After the time lapse is complete, select a new field of cells and repeat the process. Begin by determining the fluorescence intensity of a background region outside of the cell to be used for background correction.
Draw a micron-sized square region outside of the cell and make a mask. To determine the fluorescence intensity, click on Analyze, Mask Statistics. Select Mean fluorescence intensity and export the values.
Determine the fluorescence intensity within the photoactivated region for each time point. Select Mask to highlight the region that was photoactivated. To determine the fluorescence intensity, click on Analyze, Mask Statistics.
Select Mean fluorescence intensity and Export the values. Obtain the X and Y coordinates of the center of the photoactivated region by selecting Mask to highlight the region that was photoactivated. Once the region of photoactivation is highlighted, select Analyze, Mask Statistics, Center of Area, and Export the values.
Determine the X and Y coordinates of the fluorescent probe of interest. Select Manual Particle Tracking and click on the fluorescent probe of interest over time. Once all of the time points have been tracked, select Analyze, Mask Statistics, Center of Area, and Export the values.
T cells were attached to chambered cover glass containing photoactivatable MCC-IEFK. C1-GFP, a probe for the lipid second messenger DAG, was imaged using TIRF microscopy and RFP-Tubulin in epifluorescence mode. Localized photoactivation of the surface beneath the T cell induces the accumulation of DAG in the irradiated zone.
This is followed within seconds by the reorientation of the centrosome to the same region. C1-GFP accumulation can be quantified by calculating the normalized fluorescence intensity after background correction at the center of the photoactivated region over time. Centrosome reorientation in response to photoactivation can be quantified by calculating the distance between the centrosome and the center of the photoactivated region as a function of time.
Once mastered, this technique can be done in less than a day if it is performed properly. We typically generate 15 to 20 time lapse movies for analysis in this time. This approach paved the way for researchers to explore the precise kinetics and polarization of activating signaling in T cells.
It was also adapted to study inhibitory signaling in natural killer cells. As a result, we now have a better understanding of how communicative immune cell cell interactions are assembled and dissolved.
