Cytotoxic T Lymphocyte Killing Assays

CD8⁺ cytotoxic T lymphocytes (CTL) are important effector cells of the immune system.  By destroying cells that express foreign antigens on their surface through MHC class I molecules they protect against viral infections and also against certain cancers.

⁵¹Chromium (Cr) Release Assays

The classical assay to detect CTL activity is the ⁵¹Cr release assay.  Essentially this assay assesses the ability of CD8⁺ cytotoxic T cells to lyze target cells expressing an epitope of interest.

• Target cells are stimulated with an appropriate peptide to activate the cells

• Target cells expressing the epitope of interest are labeled with ⁵¹Cr

• Cells are incubated with CTL effector cells

• As the CTL effector cells bind to the antigen specific target, the cells are lyzed and the ⁵¹Cr is released into the culture supernatant

• The cells are centrifuged and the level of ⁵¹Cr in the supernatant is measured by liquid scintillation.

The main drawback to using ⁵¹Cr release assays is the involvement of radioactive reagents which requires specialist laboratory certification and specially trained users.  In addition the protocol can only be carried out on fresh cells; cryo-preserved cells cannot be used.

Alternative Killing Assays

In recent years there have been a variety of other CTL assays developed that use non-radioactive reagents.  Several of these involve measurement of caspase 3 cleavage in apoptotic cells to determine the levels of CD8⁺ cytotoxic activity^1,2 and can be adapted for either flow cytometry or immunofluorescent staining.  These methods are proving to be more sensitive than current ⁵¹Cr release protocols and can provide equivocal quantitative data to ELISPOT assays and MHC multimer staining.

References

¹Jerome KR et al. (2003) Measurement of CTL-induced cytotoxicity: The caspase 3 assay. Apoptosis, 8: 563-571 [PubMedID: 14574062]

²He L et al. (2005) A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. J Immunological Methods, 304: 43-59 [PubMedID: 16076473]

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