An Assay to Evaluate Immunodominance in Cytotoxic T Lymphocyte Responses
Transcrição
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.
