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Abstract：
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.
摘要：
需要新的(免疫)疗法来稳定AML中的缓解或疾病。白血病衍生的树突状细胞(DCleu)可以使用批准的药物(GM-CSF和PGE-1(KitM))从AML患者的全血中离体产生。T细胞富集后，KitM预处理的混合淋巴细胞培养(MLC)(vs.未经处理的WB)，适应性和先天免疫系统的抗白血病免疫细胞已经显示显著增加.我们评估了（1）白血病特异性测定的使用[INFy的细胞内细胞因子产生，TNFa(INCYT),和通过CD107a（DEG）检测的脱颗粒]用于详细定量白血病特异性细胞和（2），此外,KitM治疗与功能性细胞毒性和患者临床数据的相关性未预处理设置。我们在首次诊断时收集了26名AML患者的全血（WB）样本，在持续的疾病期间，或同种异体干细胞移植（SCT）后复发，来自18名健康志愿者。WB样品用或不用试剂盒M处理以产生DC/DCleu。用KitM处理后的MLC与未经治疗的WB抗原特异性/抗白血病作用通过INCYT评估，DEG,和细胞毒性氟解试验。通过流式细胞术进行细胞亚型的定量。我们的研究表明：（1）在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KitM处理的vs.中未诱导母细胞增殖的情况下，（成熟）DCleu的产生频率明显更高未经处理的样品。（3）免疫反应性细胞的频率显着增加（例如，非幼稚T细胞，Tprol）以及INCYT/DEG评估白血病特异性适应性（例如，B,T（记忆））或先天免疫细胞（例如，NK,CIK)用KitM处理后的MLC与未经处理的WB。细胞内产生INFy和TNFa的结果是相当的。细胞毒性氟解测定显示，在KitM处理的细胞中，与未经处理的WB。在来自几个细胞系的诱导的白血病特异性细胞和改善的母细胞裂解之间可以显示出显著的相关性。我们使用功能测定（DEG，INCYT,和CTX）。我们可以量化未培养的WB以及MLC后的白血病特异性亚型，并评估KitM预处理（含DC/DCleu）WB对提供白血病特异性免疫细胞的影响。KitM预处理(vs.无预处理）显着增加白血病特异性IFNy和TNFa的产生，去粒化细胞并改善MLC后的原始细胞毒性。用KitM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。INCYT和DEG测定有资格在单细胞水平上定量潜在的白血病特异性细胞并预测接受治疗的患者的临床过程。
