The overall goal of the following experiment is to determine cytotoxic T-cell activity by comparing an impedance based approach using the EXOGEN system to the chromium release assay. The first step is achieved by separating peripheral blood mononuclear cells from whole blood using fial. Next, the PBMC are added to wells along with peptide antigen, human recombinant IL two, and irradiated PBMC to create antigen-specific CD eight T cells.
The antigen specific CD eight T cells are then co cultured with tumor cells in both assays in order to cause tumor cell death. Results are obtained that show percent lysis of tumor cells using both the impedance method and the chromium release assay. The main advantage of this technique over existing methods like the chromium release assay, is that the impedance based system collects real-time data and requires less cells that don't need any labeling For this protocol, blood from normal healthy HLA A two positive donors provides a source of pbmc or peripheral blood mononuclear cells following the manufacturer's protocol.
Use F call PA plus to separate the P BMCs from the blood. Next into six well plates load approximately 4 million P BMCs in two milliliters of media per well. To each, well add HLAA two binding peptide for a final concentration of 10 micrograms per milliliter culture.
The cells on alternate days to help stimulate and expand T-cells. Add fresh media supplemented with human recombinant IL two for a final concentration of 50 units per milliliter. After one week, prepare a culture of autologous pbmc, irradiate the cells and pulse them for two hours with the same hla.
A two binding peptide at 10 micrograms per milliliter restimulate the culture by adding one milliliter of the irradiated autologous PBM CS to each. Well continue adding fresh media supplemented with IL two on alternate days after another week, restimulate the cultures in the same manner a second time. After six more days in culture, the cells will be ready for use.
Prepare the intelligence impedance measuring station for use by equilibrating it to 37 degrees Celsius under 5%carbon dioxide for at least an hour. Harvest 75%confluent human tumor cells using 0.25%trypsin and centrifugation. Count the tumor cells using a hemo cytometer and reconstitute them in RPMI.
Tumor media at a concentration of 7, 500 cells per microliter. Next program, the Excel agent software set up the layout page with the well layout. Set up the schedule page to take impedance readings every five minutes for a 40 hour period for the first 18 hours, the Xceligent system will only take impedance readings of the tumor cells.
Then add 100 microliters of RPMI tumor media per well. Take a background reading on thence EPLs by measuring impedance in the absence of cells. After the tumor cells are added, load the plates in thence station and start taking readings.
The tumor cells will immediately start adhering to the EPLs. After about 18 hours of incubation, the tumor cells will have attached to the EPLs and doubled to 15, 000 tumor cells per well. At this time, harvest prepared T cells by gentle scraping and agitation centrifuge.
The T cells, reconstitute them in RPMI media with 10%FBS and count them using a hemo cytometer. Once the cell density is known, prepare a twofold dilution series of T cells such that when 200 microliters of T cells are added to the tumor cells, the highest concentration is 40 T cells per tumor cell. The last dilution of the series should provide 1.25 T cells per tumor cell.
Pause the EXOGEN station and remove the EPL using a pipette. Remove the media in the wells, then load 200 microliters of T cells using all the concentrations from the dilution series as a negative control. Use pure media, return the EPL to thence station and continue the program at the end of the assay.
Normalize the results. Always follow institutional radiation safety procedures when working with chromium 51 and chromium 51 labeled target cells and use the shielding to reduce radiation exposure. Begin by pulsing tumor cells for two hours at 1 million cells per milliliter with 10 microliters per milliliter of fresh chromium 51.
Next, wash the tumor cells in 10 milliliters of RPMI media with 10%FBS centrifuge the cells and reconstitute them in the same media at 1 million cells per milliliter. Then to a 96 well round bottom plate. Add 100, 000 tumor cells per well to calculate spontaneous release.
Do not add anything else to six of these wells to six other wells. Add 100 microliters Triton X 100 to lyce the cells and hence calculate maximum release to all the remaining wells. Add T-cells to the plate using the dilution series prepared previously.
Now incubate the plates for five hours and collect 50 microliter samples of supernatant from each, well load the samples onto a luma plate after drying the plates in the hood overnight at room temperature. Measure their CPM using a gamma counter T-cell and SKBR three. Cancer cells were cultured on X intelligence plates and the cell index was measured, which is a reflection of the plate impedance because T cells do not require adherence to proliferate.
Their effect on impedance was negligible over a 40 hour culture. Cancer cells cultured alone showed an increasing impedance measurement. However, an addition of T cells at 18 hours disrupts this trend.
This event requires software normalization after the addition of T cells and is further investigated in the next experiment. When SK BR three cells were co cultured with varying concentrations of T cells, reductions in impedance were very clearly dependent on the dose of T-cell data was collected every five minutes over three trials at the 10 hour mark. Cell indices followed a second order polynomial over the range of T cells indicating that the Exogen station monitors CD eight T-cell mediated death of SK B three tumors in real time as a drop in cell index to determine if reductions in impedance of SK BR three by the T cells were antigen specific.
SK KBR three cells were co-culture with HLA A two positive FLU specific T cells that do not recognize HER two new. These cells had significantly less lytic activity to further determine if her two new specific T cells were killing in an antigen specific fashion antibody to fast ligand was incorporated into the co cultures. The antibody did not reduce the lytic activity of the HER two new P 360 9 specific T cells.
However, with T cells that were non-specifically activated with anti CD three, CD 28 beads, the antibody did inhibit lysis to determine if the killing was occurring in an HLA class one dependent manner. Either anti HLA class one antibody or isotype control antibody were added to the co cultures. The results strongly supported this hypothesis.
T cells were next co cultured with other cancer cells at a one to five ratio. The HLA A two negative tumor cell line BT 20 was used as a negative control. The results show that the method is useful for multiple target adherent tumor cells.
Co cultures of the HER two new P 360 9 specific T cells were added to chromium 51 labeled target cells, followed by CRA analysis. The intelligence assay appears more sensitive to analyze consistency. Intra assay variation was calculated as the percent coefficient of variation at each cell ratio between one to 40 and one to five.
This measured below 15%at the lowest cell ratios. However, the percent CV was high or unpredictable. Additionally, the variability of SK BR three T-cell lysis by co-culture with different P 360 9 specific T-cell lines was examined.
Data was generated 30 days apart. The assays were performed in triplicate and the results were very similar. After watching this video, you should have a good understanding of how to determine cytotoxic T-cell activity using antigen specific CD eight T cells and tumor cells utilizing an impedance based approach.
