CD14+单核细胞系白血病细胞来源树突细胞体外刺激特异抗白血病T细胞应答

背景与目的:白血病细胞能在体外分化为树突细胞(dendriticcells,DCs),从而有希望用于白血病的免疫治疗。本研究旨在探讨CD14高表达的单核细胞系白血病(M4、M5)细胞分化而来的DCs体外诱导抗白血病T细胞应答的能力。方法:取5例初诊CD14高表达的M4或M5型白血病患者的骨髓标本,分离单个核细胞(bonemarrowmononuclearcells,BMMNCs),将白血病细胞分为3组:贴壁白血病细胞组、非贴壁白血病细胞组及总白血病细胞组。流式细胞术(flowcytometry,FCM)比较3组细胞的CD14表达。用含GM-CSF、IL-4和TNF-α或不含细胞因子的培养液培养细胞7～10天后,通过细胞形态学观察及FCM检测细胞表型,鉴定单核白血病细胞来源的DCs(monocyticleukemiacell-deriveddendriticcells,Mo-LDCs);采用异基因混合淋巴细胞反应(allogeneicmixedlymphocytereaction,Allo-MLR)以及细胞毒性T淋巴细胞(cytotoxicTlymphocytes,CTL)抗白血病细胞毒分析检测Mo-LDCs的免疫功能,染色体核型分析结合异常表面抗原确定Mo-LDCs的白血病来源。结果:3组中贴壁白血病细胞的CD14含量最高,在细胞因子联合诱导下,可分化为大量CD83 成熟DCs。在同一病例的3组细胞以及不同病例的总单核细胞组间,培养前CD14的表达率与诱导后CD83 DCs的产率成正相关(r=0.967,P=0.007)。Mo-LDCs具有典型的成熟DCs的形态及表型特征,在Allo-MLR中能刺激同种T细胞明显增殖,并能刺激扩增特异性抗白血病CTL。同时,Mo-LDCs持续存在所起源白血病的核型异常和异常表达的髓系抗原。结论:在细胞因子组合诱导下,M4、M5亚型AML的CD14 细胞可分化为具有免疫功能的Mo-LDCs,单核系白血病细胞的CD14表达高低可能预示其DCs分化能力。Mo-LDCs具有经典的DCs的表型及功能,还具有白血病的克隆异常,可用于M4、M5患者的免疫治疗。

In vitro stimulation of specific antileukemia T-cell response by dendritic cells derived from CD14+ acute monocytic leukemia cells

BACKGROUND & OBJECTIVE: Dendritic cells (DCs) or DC-like cells had been successfully induced in vitro from leukemia cells, which may provide a promising immunotherapeutic protocol for leukemia. This study was designed to investigate the efficiency of in vitro generation of dendritic cells from CD14+ acute myelomonocytic (M4) or monocytic (M5) leukemia cells and their ability of stimulating specific antileukemia T-cell response. METHODS: Bone marrow mononuclear cells (BMMNCs) were isolated from 5 M4/M5 leukemia patients with high CD14 expression, and then divided into 3 groups: adherent leukemia cells, nonadherent blasts, and total unfractioned blasts. CD14 expression of the 3 groups was evaluated by flow cytometry (FCM). When cultured with or without granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) for 7-10 days, monocytic leukemia cell-derived dendritic cells (Mo-LDCs) were identified through morphologic observation and immunophenotype analysis using FCM. The immune function of Mo-LDCs was detected through allogeneic mixed lymphocyte reaction (Allo-MLR) and cytotoxicity assay of antileukemia cytotoxic T lymphocytes (CTLs). The leukemic origin of Mo-LDCs was confirmed by chromosomal karyotype analysis combined with the aberrant expression of myeloid antigens. RESULTS: The amount of CD14+ cells, which could differentiate into CD83+ mature DCs under induction of the cytokine combination, was higher in adherent leukemia cells than in nonadherent blasts and total unfractioned blasts. Regarding each 3 cell groups of the same patient or the unfractioned blasts of various patients, initial CD14 expression was positively related to the yield of CD83+ DCs after induction (r = 0.967, P = 0.007). Mo-LDCs exhibited typical morphology and phenotype as mature DCs, induced potent proliferation of homogeneous T cells in Allo-MLR, stimulated the expansion of leukemia-specific CTLs, and continued to possess the cytogenetic abnormalities of the original leukemia, as well as the aberrant expression of myeloid antigens. CONCLUSIONS: In M4/M5 subtype of AML, CD14+ cells could differentiate into immune-competent Mo-LDCs under the induction of the cytokine combination. CD14 expression level may predict the DCs differentiation ability of monocytic leukemia. Mo-LDCs, which possess the classical phenotype and function of DCs, as well as the abnormal leukemic antigens, may be useful for the immunotherapy of M4/M5 AML.