Weidmann E, Brieger J, Jahn B, Hoelzer D, Bergmann L, Mitrou P S
Department of Internal Medicine, J. Goethe University Hospital, Frankfurt/M, Germany.
Ann Hematol. 1995 Mar;70(3):153-8. doi: 10.1007/BF01682036.
Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of 51Cr and/or spontaneously release high amounts of 51Cr. Recently, we established a culture system which promotes the outgrowth of cytotoxic T lymphocytes 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 cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20:1). However, a minority of AML 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 tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10:1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.
使用白细胞介素2免疫疗法治疗急性髓细胞白血病缓解期患者是延长该疾病缓解期的一种新方法。作为根除残留髓细胞母细胞群体病理生理学的重要机制，细胞毒性效应淋巴细胞的激活经常被讨论。然而，相关的免疫学研究在一定程度上较为复杂，因为在传统的铬51释放试验中，母细胞经常无法摄取足够量的51Cr和/或自发释放大量的51Cr。最近，我们建立了一种培养系统，可促进在白细胞介素2中培养的骨髓来源单核细胞中细胞毒性T淋巴细胞的生长。为了研究获得的T细胞系和克隆的细胞毒性及其相关机制，我们改进了先前描述的基于乳酸脱氢酶释放（LDH释放试验）的细胞毒性试验，用于检测从急性髓细胞白血病患者骨髓中获得的冻存母细胞。使用该试验，我们能够检测到来自三名健康对照的白细胞介素2激活的外周血淋巴细胞对从急性髓细胞白血病患者骨髓中获得的多个母细胞样本的细胞毒性（在效应细胞与靶细胞比例为20:1时，裂解率高达40%以上）。然而，少数急性髓细胞白血病母细胞似乎对白细胞介素2激活的淋巴细胞裂解具有抗性。在急性髓细胞白血病患者的骨髓来源T细胞系中，通过LDH释放检测，我们在七个测试案例中的三个中检测到了针对自体母细胞的裂解活性，在效应细胞与靶细胞比例为10:1时，裂解率在29%至63%之间。此外，还建立了具有不同表型的T细胞克隆，它们能够介导对自体母细胞的细胞毒性。因此，通过LDH释放试验可以可靠、可重复且无需使用同位素地分析对急性髓细胞白血病患者新鲜分离母细胞的细胞毒性。
