需要新的（免疫）疗法来稳定 AML 的缓解或疾病。白血病衍生树突状细胞 (DCleu) 可以使用批准的药物（GM-CSF 和 PGE-1 (Kit M)）从 AML 患者的全血中离体产生。在用 Kit M 预处理（与未处理的 WB 相比）进行 T 细胞富集、混合淋巴细胞培养 (MLC) 后，适应性和先天免疫系统的抗白血病定向免疫细胞已显示显着增加。我们评估了 (1) 使用白血病特异性检测 [INFy、TNFa (INCYT) 的细胞内细胞因子产生和 CD107a (DEG) 检测的脱颗粒] 对白血病特异性细胞进行详细定量，以及 (2) 此外， Kit M 治疗组与未治疗组的功能性细胞毒性和患者临床数据的相关性。我们从 26 名首次诊断时、疾病持续期间或同种异体干细胞移植 (SCT) 后复发时的 AML 患者以及 18 名健康志愿者中收集了全血 (WB) 样本。使用或不使用 Kit M 处理 WB 样品以生成 DC/DCleu。使用 Kit M 处理的 MLC 与未处理的 WB 抗原特异性/抗白血病效果通过 INCYT、DEG 和细胞毒性荧光溶解测定进行评估。通过流式细胞术进行细胞亚型的量化。我们的研究表明：(1) AML 患者血液中可检测到的白血病特异性细胞（亚型）的频率较低。 (2) 在 Kit M 处理的样品中，与未处理的样品相比，在不诱导母细胞增殖的情况下，可生成（成熟）DCleu 的频率显着更高。 (3) 免疫反应细胞（例如，非初始 T 细胞、Tprol）以及 INCYT/DEG 检测中白血病特异性适应性细胞（例如 B、T（记忆））或先天免疫细胞（例如 Tprol）的频率显着增加。 、NK、CIK）在使用 Kit M 处理的 MLC 后与未处理的 WB 进行比较。 INFy和TNFa的细胞内产生结果具有可比性。细胞毒性荧光裂解测定显示，与未处理的 WB 相比，Kit M 处理的细胞裂解显着增强。来自多个细胞系的诱导白血病特异性细胞与改善的胚细胞裂解之间可以显示出显着的相关性。我们使用功能测定（DEG、INCYT 和 CTX）成功地在单细胞水平上检测和定量了免疫反应性细胞。我们可以量化未培养的 WB 中以及 MLC 后的白血病特异性亚型，并评估 Kit M 预处理（含 DC/DCleu）WB 对提供白血病特异性免疫细胞的影响。 Kit M 预处理（与无预处理相比）可显着增加白血病特异性 IFNy 和 TNFa 的产生、脱颗粒细胞，并改善 MLC 后的母细胞毒性。使用 Kit M 对 AML 患者进行体内治疗可能会产生抗白血病作用，并有助于稳定疾病或缓解病情。 INCYT 和 DEG 检测有资格在单细胞水平上量化潜在的白血病特异性细胞，并预测接受治疗的患者的临床病程。

Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients' blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients' clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients' blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive-(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.