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Characterization of a granule-independent lytic mechanism used by CTL hybridomas
R Garner1, C D Helgason, E A Atkinson
1Department of Biochemistry, University of Alberta, Edmonton, Canada.
Abstract:
The mechanism(s) by which CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granzymes) contained within the granules of CTL and NK, have been implicated, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of killing used by two cytolytic hybridomas (PMM-1 and MD90) that express neither perforin nor the granzymes. These characteristics are compared with results obtained by using a representative Ag-dependent, granule-containing T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target cells in the presence of cyclosporin and the absence of calcium. All the effectors could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the target cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent on protein synthesis, and inhibited by the addition of cyclosporin. Although TNF was shown not be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the level of expression of Fas Ag. With the use of L1210 and an L1210 cell line transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effectively inhibited with anti-Fas Ab. Furthermore the lack of sensitivity to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.
Insights
Cytotoxic T lymphocytes (CTL) can kill target cells via perforin-independent pathways. This study shows two CTL hybridomas utilize the Fas pathway for target cell lysis, independent of perforin and granzymes.
Area of Science:
- Immunology
- Cell Biology
Background:
- Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells induce target cell lysis through various mechanisms.
- While perforin and granzymes are well-established cytotoxic mediators, alternative lytic pathways are increasingly recognized.
Purpose of the Study:
- To characterize the cytotoxic mechanism of two perforin- and granzyme-deficient cytolytic hybridomas (PMM-1 and MD90).
- To compare their killing mechanisms with a conventional granule-containing T cell clone.
Main Methods:
- Cytolysis assays using granule-negative hybridomas and a T cell clone.
- Assessing lysis in the presence of cyclosporin, absence of calcium, and protein synthesis inhibitors (cycloheximide, emetine).
- Investigating the role of Fas ligand and Fas receptor interactions using Fas-transfected cell lines and anti-Fas antibodies.
Main Results:
- Granule-negative hybridomas lysed targets independently of calcium and in the presence of cyclosporin.
- Activation of these hybridomas required calcium and protein synthesis, and was inhibited by cyclosporin.
- Target cell sensitivity to lysis correlated with Fas expression, and lysis was blocked by anti-Fas antibodies, confirming Fas pathway involvement.
Conclusions:
- The cytolytic hybridomas PMM-1 and MD90 utilize the Fas lytic pathway for target cell destruction.
- This Fas-mediated cytotoxicity operates independently of perforin and granzymes.
- These findings highlight the significance of alternative CTL-mediated killing mechanisms.