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The role of the Fas lytic pathway in a perforin-less CTL hybridoma
Abstract:
The murine CTL hybridoma PMMI has been shown by the most sensitive techniques to be devoid of perforin. We thus used PMMI activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CTLs in the absence of perforin. We found that PMMI is equipped with membrane TNF-alpha as a potential lytic mechanism, but TNF-alpha is unlikely to be involved in acute (4 h) lytic reactions. On the other hand, PMMI readily lyses target cells expressing the gene for the Fas Ag, but does not lyse target cells expressing fas antisense DNA. The generation of fas-dependent lysis required protein synthesis in PMMI, but target cell protein synthesis was not required for lysis. Lysis of Fas-positive target cells by PMMI was accompanied by DNA fragmentation, and both lysis and DNA fragmentation were blocked by inhibition of protein synthesis in the effector cell. We find the relative extent and kinetics of fas-dependent lysis and DNA fragmentation indistinguishable from that seen in "classical" CTL lytic assays. Both fas- and perforin-dependent lysis were blocked by inhibitors of poly(ADP) ribosylation. We found very little difference in the sequence of events in target cells lysed by the fas pathway compared with the classical (probably perforin) lytic pathway. Given the widespread distribution of fas, particularly in hematopoietic target cells, caution may be required in interpreting the relationship between parameters such as DNA fragmentation and 51Cr-release solely on the basis of the granule exocytosis model.
Insights
Cytotoxic T lymphocytes (CTLs) lacking perforin can still lyse target cells. This study reveals Fas-dependent lysis as an alternative pathway, involving DNA fragmentation and protein synthesis in effector cells.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Biology
Background:
- Cytotoxic T lymphocytes (CTLs) are crucial for adaptive immunity.
- The canonical CTL-mediated lysis involves perforin-dependent granule exocytosis.
- The PMMI murine CTL hybridoma lacks perforin, necessitating investigation of alternative cytotoxic mechanisms.
Purpose of the Study:
- To investigate alternative cytotoxic pathways in perforin-deficient CTLs.
- To elucidate the role of Fas-mediated apoptosis in CTL-induced target cell lysis.
- To compare the kinetics and molecular events of Fas-dependent lysis with perforin-dependent lysis.
Main Methods:
- Utilized the perforin-deficient PMMI CTL hybridoma.
- Activated PMMI cells with PMA and ionomycin.
- Assessed target cell lysis using Fas Ag expression and fas antisense DNA.
- Investigated the requirement for protein synthesis in effector and target cells.
- Analyzed DNA fragmentation in target cells.
- Examined the effect of poly(ADP) ribosylation inhibitors on lysis.
Main Results:
- PMMI cells exhibit Fas-dependent lysis of target cells, independent of perforin.
- Fas-mediated lysis requires effector cell protein synthesis but not target cell protein synthesis.
- Lysis is accompanied by DNA fragmentation, mirroring classical CTL assays.
- Both Fas- and perforin-dependent lysis are inhibited by poly(ADP) ribosylation inhibitors.
- TNF-alpha is present but not involved in acute lysis.
Conclusions:
- Perforin-deficient CTLs can mediate target cell lysis through a Fas-dependent pathway.
- Fas-mediated cytotoxicity shares similarities in kinetics and molecular events with perforin-dependent lysis.
- The findings challenge the sole reliance on the granule exocytosis model for interpreting CTL-mediated cytotoxicity, especially given the broad expression of Fas.