A modified cytotoxicity assay with high sensitivity

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Abstract
Since adoptive immunotherapy using lymphokine activated killer (LAK) cells and interleukin-2 (IL-2) has been introduced into clinical medicine, there has been a growing interest in cytotoxicity assays. The standard 51chromium release assay has certain disadvantages, in particular limited sensitivity, because of a high, nonspecific background release. We examined the conditions under which tritiated thymidine, which has been used to assess slow macrophage mediated cytolysis, can be utilized to assess the rapid cytotoxic activity of unstimulated PBL and LAK cells. The optimal assay duration for the 3H-thymidine (3H-TdR) release assay is 24 h. Under standard conditions actively proliferating effector cells do incorporate some of the 3H-TdR released by the target cells during this time, leading to false low results. This problem can be abolished by the addition of excessive amounts of cold TdR to the assay medium. We found a good correlation of the results of the TdR release assay and the Cr release assay. Using the very sensitive TdR release assay, unexpected significant cytolysis of the so-called ‘NK resistant’ Daudi cells by unstimulated PBL is demonstrated. The modified 3H-TdR release assay is well-suited to monitor the immunological effects of immunotherapy, using IL-2 and LAK cells.