Technical noteA novel fluorescent sensitive assay for detection of differential T cell mediated lysis of multiple adherent target cells
Introduction
Cytotoxic T cells (CTL) are a major component of the adaptive immune system. Their ability to selectively lyse target cells presenting foreign antigen protects against intracellular pathogens such as bacteria, protozoa and viruses in addition to having a role in tumour and allogenic graft rejection (Zinkernagel, 1996). A number of assays have been used to assess the cytolytic activity of CTL, including release of chromium from labelled targets (Brunner et al., 1968), detection of caspase 3 induction (Liu et al., 2002) and a flow cytometry based assay for measuring loss of fluorescently labelled targets (Sheehy et al., 2001). These valuable approaches use suspensions of target cells, which if the targets are an adherent cell type, require trypsinisation or calcium chelation prior to use. Whilst in many settings such treatment may not alter the presenting capacity of adherent targets cells, we wished to use a technique for which we could exclude such potential artefact. However, there have been surprisingly few investigations that have used adherent cells as CTL targets. Several studies have used adherent target cell detachment as a marker for interaction with T cells, but target cell lysis was dissociated from target cell detachment (Russell et al., 1988, Abrams and Russell, 1991, Wang et al., 2004) suggesting that whilst the two processes are linked they are nevertheless distinct events. We therefore wished to develop a reproducible sensitive quantitative assay of adherent cell death by T cells, with the capacity to investigate differential killing of multiple targets at the same time.
Section snippets
Effector cells
Peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation. All samples were taken from consenting healthy adults under ethical approval from the Oxfordshire Ethics Committee. Cells were suspended in RPMI 1640 plus 10% human AB serum for generation of specific CD8+ T cell lines. The PBMC were incubated with 100 μM EBV BMLF1 280-8 GLCTLVAML peptide which was synthesized using standard Fmoc chemistry. Peptide purity was
Fluorescent labelling of keratinocytes
Keratinocytes were treated with 300 U/ml IFN-γ (Roche) overnight. IFN-γ treatment was necessary to sensitise keratinocytes to T cell mediated cytotoxicity (Symington and Santos, 1991). The keratinocytes were then trypsinised and incubated with 5 μM CFSE (carboxy-fluorescein succinimidyl ester; Invitrogen) or CellTracker Orange CMTMR ((5-(and 6)-(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine; Invitrogen) diluted in PBS 0.05% bovine serum albumin for 10 min at 37 °C. Both CFSE and CMTMR
Acknowledgements
We are most grateful to the MRC for funding support and to the volunteers who kindly donated blood samples. HLA-A2 positive keratinocytes were a gift from Dr E. O'Toole and Dr. N. Baksh.
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