An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses

Begin with fluorescent dye-labeled target cells presenting a non-reactive peptide epitope or tumor antigen-derived peptide epitopes with varying immunogenicity.

These cells exhibit varying fluorescence intensities — low, intermediate, and high — corresponding to the presentation of non-reactive, subdominant, and immunodominant peptide epitopes, respectively.

Administer the cell suspension intravenously to a sensitized mouse.

The injected cells travel to the spleen containing immune cell populations, including cytotoxic T lymphocytes or CTLs.

Within the spleen, CTLs interact with the epitopes of target cells.

Immunodominant epitopes, being more immunogenic, trigger a robust CTL immune response, releasing cytotoxic substances and resulting in high-intensity fluorescent cell lysis.

Collect the mouse spleen and mechanically disrupt it. Add a lysis buffer to eliminate red blood cells and filter the suspension.

Centrifuge and resuspend the cell pellet.

Sort the cells using a fluorescence-activated cell sorter. The reduction in high-intensity fluorescent cells, compared to intermediate and low-intensity fluorescent cells, confirms immunodominance in the cytotoxic T lymphocyte response.

For injection of the target cells, first, gently vortex the source tubes and pool the three CFSE-labeled cell suspensions into a new tube at equal ratios. Top up the tube contents with sterile PBS, and collect the cells by centrifugation for counting. Then, adjust the volume to a 1 x 10⁷ mixed target cells per 200 microliters of PBS per recipient concentration, and inject 200 microliter volumes of the cell suspension into the tail vein of each recipient C57-Black-6 mouse.

Two or four hours after the injection, remove and process the spleen of each recipient animal as demonstrated, and resuspend the isolated white blood cells in 3 milliliters of PBS per spleen. Transfer approximately 1 times 10 to the 7th cells from each processed spleen into a clean FACS tube, and immediately run the cells on the flow cytometer as demonstrated to gate the CFSE low, intermediate, and high target cell populations.

Then, acquire a total of 2,000 CFSE low events in the fluorescence one channel, and calculate the specific lysis of each cognate target cell population, according to the formula detailed in the text.