Use of a P815‐derived line with an amplified adenosine deaminase gene: an improved target for cellular cytotoxicity

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Abstract
We describe a cytotoxic T lymphocyte‐mediated cytotoxicity assay in which the release of a cytoplasmic enzyme, adenosine deaminase (ADA), instead of the widely used radioactive chromium is a measure of target lysis. In this enzyme‐release assay the target is a mastocytoma P815‐derived cell line, noted P815 ADA++, isolated by applying a selection procedure devised to specifically amplify the ADA gene. Gene amplification in P815 ADA++ was indeed demonstrated. Routine measurement of ADA activity from numerous supernatants is performed using a specific and sensitive colorimetric assay. The use of 96‐well microtiter plates as well as of an automatic Multiscan spectrophotometer makes this measurement rapid and convenient. We show that this ADA‐release assay is significantly more sensitive than the classical chromium‐release test because of its consistently lower (5 to 10‐fold) spontaneous release in 4 h, short‐term cytotoxicity experiments. We also found that it is especially suited for the rapid detection, by visual screening, of rare, active killer clones among large, heterogeneous cytotoxic T lymphocyte populations. The assay could easily be adapted to other tumor targets (EL4, YAC‐1, K562) of common use in studies involving immune lysis; indeed, the procedure of amplifying the ADA gene used in the isolation of the P815 ADA++ hyperactive line may be generally applied to these targets.