LACTATE DEHYDROGENASE-RELEASE ASSAY - A RELIABLE, NONRADIOACTIVE TECHNIQUE FOR ANALYSIS OF CYTOTOXIC LYMPHOCYTE-MEDIATED LYTIC ACTIVITY AGAINST BLASTS FROM ACUTE MYELOCYTIC-LEUKEMIA

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remis sion duration in this disease. As an important mechanism for the patho physiology of eradication of residual myelocytic blast populations, ac tivation of cytotoxic effector lymphocytes has frequently been discuss ed. However, the associated immunological research has been complicate d to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of Cr-51 and/or spontaneously release high amounts of Cr-51. Recently, we esta blished a culture system which promotes the outgrowth of cytotoxic T l ymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T -cell lines and clones, we modified a previously described cytotoxicit y assay, based on the release of lactate dehydrogenase (LDH-release as say) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were abl e to detect cytotoxicity of IL-2-activated peripheral blood lymphocyte s from three healthy controls against a number of blast samples obtain ed from the bone marrow of patients with AML (up to more than 40% lysi s at an effector target cell ratio of 20:1). However, a minority of AM L blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases test ed by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were es tablished which were able to mediate cytotoxicity against autologous b last cells. Thus, cytotoxicity against freshly isolated blasts from pa tients with acute myelocytic leukemia can be analyzed reliably, reprod ucibly, and without the use of isotopes by the LDH-release assay.