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Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing

Abstract

Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli β-galactosidase in mouse or human target cells, and an Elispot to detect release of β-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-γ secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

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Figure 1: The Lysispot assay measures the frequency of antigen-specific, perforin-dependent T-cell killers.
Figure 2: Measurement of peptide-specific human cytolytic T-cell frequencies by Lysispot.
Figure 3: Relationship of bulk lysis cytotoxicity assays to frequency measurement by Lysispot.
Figure 4: IFNγ secretion and cytotoxicity by CD8 T cells can be independently regulated.

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Acknowledgements

We thank Wade Narrow for technical assistance. This work was supported by the National Institute for Allergy and Infectious Disease (R01 AI48414).

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Correspondence to Tim R. Mosmann.

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Snyder, J., Bowers, W., Livingstone, A. et al. Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. Nat Med 9, 231–236 (2003). https://doi.org/10.1038/nm821

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