Caught in the act

Microscope images of CTL-mediated killing of target cells detected using the new fluorescence-based assay. Reproduced with permission © (2002) Macmillan Magazines Ltd.

Peptide–MHC-class I tetramers have revolutionized our understanding of T-cell biology by allowing us to track antigen-specific CD8⁺ T cells (also known as cytotoxic T lymphocytes, CTLs) during the course of an immune response. But does specificity always equate with function? To answer this, tetramers must be used in conjunction with assays of activity; however, currently only one arm of CTL function — cytokine production — can be assessed at the level of the single cell. But now, Luzheng Liu and co-workers at the Emory Vaccine Center have developed a new assay that allows cytotoxic killing to be detected at the single-cell level, in real time.

CTLs induce apoptosis of their targets, a process that takes several hours. The traditional CTL assay measures the bulk release of radioactivity from ⁵¹Cr-labelled target cells when they eventually lyse — the end point of the cytotoxic process in vitro. But the new assay, which is described in the February issue of Nature Medicine, is based on the detection of a very early event in the induction of apoptosis — the activation of caspases.

In the new assay, effectors are incubated with target cells for several hours then a cell-permeable fluorogenic caspase substrate is added. Each synthetic substrate consists of a peptide conjugated to two fluorophores, but the close proximity of the fluorophores quenches fluorescence. When the substrates are cleaved by active cell caspases the products fluoresce and so the condemned target cell can be detected by flow cytometry and microscopy.

But how does the sensitivity of the new CTL assay compare with the ⁵¹Cr-release assay? The authors performed the two assays in parallel using splenocytes from mice infected with lymphocytic choriomeningitis virus (LCMV) as effectors and targets pulsed with various LCMV peptides. The fluorescence-based assay was shown to be more sensitive than the ⁵¹Cr-release assay, particularly at earlier time points.

A key advantage of the new assay is that the interaction between the target and CTL leading to the delivery of the lethal hit can be visualized directly, in real time, by fluorescence microscopy. In combination with peptide–MHC tetramer staining, this powerful new technique should lead to new insights into the killing process, as well as more accurate measurements of CTL responses.