Abstract
Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).
Keywords: CD8 T cell, Cytotoxic assay, Tumor killing assay, Chromium-51, Functional avidity
Materials and Reagents
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Acknowledgments
This work was supported by the Department of Oncology of the University of Lausanne and the Ludwig Center for Cancer Research of the University of Lausanne. The Chromium Release Assay has been developed by researchers of the Swiss Institute of Experimental Cancer Research (ISREC) and the Department of Biochemistry at the University of Lausanne (Switzerland) and was first published in 1968 (see Reference 1).
References
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