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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 activity1,2 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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