One of the best ways to study Fas-based cytotoxicity in the absence of the perforin/granzyme contribution is using cytolytic effector cells that are defective in the perforin/granzyme system, while keeping intact their ability to induce FasL expression. This was first done in the laboratory of Dr Pierre Golstein (Marseille, France) by serial subcloning from the cytolytic hybridoma PC60 to generate dlOS, dllS, and dl2S cells (2). These cells were able to kill Fas-expressing cells once activated with a combination of PMA plus ionomycin whether in the presence or absence of extracellular calcium. They were later used in the laboratory of Dr Shikegazu Nagata (Osaka, Japan) to clone FasL (3).
Another approach has been to isolate effector cells from perforin knockout mice. In these mice, the whole perforin/granzyme system is defective, while FasL expression is normal (1,4).
However, normal effector cells, harbouring all the cytotoxicity mechanisms mentioned, can also be used to study Fas-based cytotoxicity in the absence of a perforin/granzyme contribution, or vice versa. This allows a good estimation of the relative contribution of each cytotoxicity mechanism in a given experimental system. We will describe suitable methods using whole functional CTL clones (13,15).
Protocol 6. Assay of Fas-based cytotoxicity in the absence of perforin/granzyme contribution
• 600 nM ionomycin
• anti-TCR or anti-CD3 antibodies
• sodium carbonate/bicarbonate buffer, pH 8.0
1. Culture CTL clones in complete culture medium supplemented with IL-2, in 24-well plates, to a density between 3 and 6 x 105 cells/ml. Then, stimulate the cells for 3 h in the same culture conditions to induce FasL expression with one of the following stimuli:
(i) a combination of PMA and ionomycin.
(ii) anti-TCR or anti-CD3 antibodies. In the case of antl-CD3 antibodies, their previous immobilization on wells of a 24-well plate by overnight incubation of a 20 n,g/ml antibody solution at 4°C in sodium carbonate/bicarbonate buffer recommended. (Mi) antigen-bearing target cells. In this case, it is recommended that a 1:0.7 effector to target ratio is used, to minimize the interference between the activating cells and the Fas-bearing target cells In the subsequent cytotoxicity assay.
2. After stimulation, wash out the stimulating agents, and test functional FasL expression in a cytotoxicity test on Fas-expressing target cells using one of the protocols described above. A good system for such an assay is the use of a cell line that does not express Fas and its corresponding Fas transfectant. For instance, the leukaemic cell line L1210 has been transfected with the Fas cDNA, generating an identical cell line which expresses high Fas membrane levels and is very sensitive to Fas-induced apoptosis (2). Then, Fas-based cytotoxicity can be assayed using any pre-stimulated effector cell on L1210Fas cells and using L1210 cells as a negative control. Any other system with such characteristics can also be chosen (e.g. L929/L929Fas).
3. To avoid any hypothetical contribution of perforin/granzymes, cytotoxicity tests on L1210 and L1210Fas cells should be performed in the absence of extracellular calcium. To do this, add EGTA and MgCI2 to the medium of the cytotoxicity test. The execution of Fas-based cytotoxicity, once FasL is expressed, is calcium independent, while the execution of perforin/granzyme-based cytotoxicity is entirely calcium dependent.
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