The overall goal of this procedure is to measure the cytotoxic activity of antigen-specific CD eight T cells using a cell-based flow cytometry assay. This is accomplished first by expanding effectors CD eight T cells for six days in the presence of the peptide of interest in the second step target CD four T cells are pulses with the peptide of interest and then mixed with unposted CD four TT cells. Next, the effector CD eight T cells and target CD four T cells are co cultured for six hours at different effector to target ratios.
In the final step, the killing of the peptide loaded target CD four T cells and the number of effector CD eight T cells are quantified by flow cytometry. Ultimately, the intrinsic killing capacity of the antigen-specific CD eight T cells expressed in lytic units can be calculated. The main advantages of this technique of oxy matter like aro release assay, is that that this technique uses autologous primary target cells instead of immortalized cell lines and the actual target cell.
That and effector cell numbers are quantifiable at the single cell level. Early now, research assistant in my laboratory will be demonstrating the procedure To isolate the CD eight positive effector cells. Begin by thawing autologous cryopreserved pbmc in a 37 degrees Celsius water bath, and then wash the cells in up to 50 milliliters of complete media.
After counting, dilute the pbmc at a concentration of 5 million cells per milliliter in complete media and then add IL two and the peptide of interest to the culture. Next seed one milliliter of the cell suspension to each well of a 96 well plate. After three days of culture, replace half of the supernatant with fresh complete media After six days of culture, use a multi-channel pipette to transfer the cells to a sterile reservoir and then count and wash the cells.
Re suspend the pellet at 50 million cells per milliliter in a separation buffer and incubate the pbmc in a 14 milliliter round bottom tube with a human CD eight positive T-cell enrichment cocktail at 50 microliters per milliliter cells at room temperature After 10 minutes, incubate the cells with anti CD eight IgG conjugated magnetic particles at 150 microliter per milliliter for five more minutes, and then bring the cell suspension up to seven milliliters with additional separation buffer. Place the tube into the separation magnet after five minutes with the tube still in the magnet, pour the cells into a 15 milliliter conical tube. Next stain a small aliquot of the cells with antibodies against CD three and CD eight in PBS supplemented with 2%FBS for 30 minutes at four degrees Celsius.
Confirm that the purity of the cells is 95%CD eight positive or higher by flow cytometry. Now add 225 microliters of complete media into five screw cap tubes, and then serially dilute the cells from one to two to one to 32. To isolate the CD four positive target cells begin by thawing autologous cryopreserved pbmc in a 37 degree Celsius water bath.
Transfer the thawed cells to a 50 milliliter conical tube containing 10 milliliters of media. Enrich for CD four expressing T cells by magnetic particle separation as just demonstrated confirming a 95%purity or higher of CD four positive T cells by flow cytometry. After counting the cells, split them into two 15 milliliter conical tubes and then wash them in warm PBS re suspend the pellets at 20 million cells per milliliter in PBS.
After ensuring the cells are protected from light stain one half of the CD four positive T cells with a freshly prepared CFSE high working solution to a final concentration of 0.2 millimolar and the other half in a freshly prepared CFSE low working solution to a final concentration of 0.04 millimolar for 15 minutes at 37 degrees Celsius in 5%CO2. At the end of the staining period, spin down the cells and resuspend the pellets in one milliliter of warm complete media to quench the labeling reaction. Pulse the CFSC low CD four positive T-cell with a final concentration of five micrograms per milliliter of the peptide of interest in complete media for 45 minutes in the cell culture incubator.
After the incubation wash both the CFSC high and low T-cell populations. Twice incomplete media and resus suspend both cell suspensions in 200, 000 cells per milliliter. Each incomplete media, then mix the two target populations at a one-to-one CFSE high to CFSE low ratio.
Now add 100 microliters of the mixed target CD four positive T cells to each well in a round bottom 96 well plate then seed one microliters of each CD eight positive T-cell suspension in duplicate for a final volume of 200 microliters per well. Measure the basal apoptosis seed three wells with target cells alone, and then incubate the co-culture for six hours in the cell culture incubator. After the incubation is complete, transfer the cells to a VBO 96 Well plate and stain each well with PE conjugated peptide MHC Tetramers for 15 minutes.
In the cell culture incubator, wash the cells with PBS supplemented with 2%FBS and then stain the cells for 30 minutes at four degrees Celsius, protected from light after washing the cells. Again, re suspend the pellets in 100 microliters of 2%from formaldehyde in PBS and analyze the cells by flow cytometry. After co-culture, the cells are analyzed and gated as demonstrated in these dot plots.
The ratio of viable CFSE high versus CFSE low target cells, unposted or peptide pulsed is analyzed from a small forward by side Scatter GA live cells are gated on live dead negative and CD four positive T cells are plotted on CFSE to analyze the ratio of CFSE low versus CFSE high cells. Next, a larger forward by side scatter gate is created to enumerate the total number of either dead or live effector and target cells. After the six hour incubation CD eight positive, tetramer positive T cells and CFSE, low CD four positive T cells are plotted to analyze the ratio of effector versus target cells.
The total number of effector CD eight positive T cells are then determined by gating on the CD eight positive tetramer positive cells. The total number of target cells is determined by gating on the CD four positive T-cells, CFSE, low and live dead positive and negative cells. The target ratio of CFSE low versus CFSE high cells is one to one.
When the CD four positive target cells are cultured in the absence of effector cells, this ratio drastically decreases. When the CFSE low positive pulse cells are incubated. With the total CD eight positive T-cell population containing antigen specific CD eight positive effector T cells.
As the ratio decreases the CFSE low CD four positive pulse target cells begin to become live dead positive Dilution of the effector cells thereby results in a restoration of the CFSE ratio and ultimately a restoration in the number of viable peptide pulses, CFSE, low CD four positive target T cells. The percentage of specific lysis can then be plotted as a function of the effector to target ratio in a log scale. As demonstrated in the graph, a linear regression is calculated from the plot and the equation of the trend line is used to calculate the lytic units.
That is the number of CD eight positive effector T cells required to kill 30%of 1 million target CD four positive T cells to determine the intrinsic cytolytic capacity of the antigen-specific CD eight positive T cells, and to compare the results between donors. After watching this video, you should have a good understanding on how to use the cytometry to measure oxic activity of antigen specific CD T cells. This technique can also be easily adopted for your own F cell type.
