Abstract
Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing forthe detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allowsthe researcher to assess the antigenspecific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may resultin differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.
Original language | English (US) |
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Article number | e56255 |
Journal | Journal of Visualized Experiments |
Volume | 2017 |
Issue number | 129 |
DOIs | |
State | Published - Nov 28 2017 |
Keywords
- Acute response
- CD8 T cells
- CFSE-labeling
- Cytolytic function
- Immunology
- In vivo assay
- Issue 129
- Peptide vaccination
ASJC Scopus subject areas
- General Neuroscience
- General Chemical Engineering
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology