Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy
Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy
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DOI：
10.1097/00000658-199707000-00002
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发表时间：
1997-07-01
影响因子：
9
通讯作者：
Smith, C
中科院分区：
文献类型：
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作者：
Morse, MA;Zhou, LJ;Smith, C
该研究的目的是表征在可接受的条件下产生大量成熟树突状细胞（DC）的前体、发育途径和培养基的要求，所述条件可用于手术治疗恶性肿瘤的辅助、主动免疫治疗策略。随着体外从外周血单核细胞（PBMC）有效产生大量DC的方法的发展，用于恶性肿瘤的基于DC的免疫疗法已经变得更加现实，但是这些方法依赖于临床上不可接受的培养条件（例如包含胎牛血清），因此需要开发在无血清条件下产生功能等同DC的方法。（来自健康供体和癌症患者）与粒细胞-巨噬细胞-集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）在有和没有肿瘤坏死因子-α的情况下孵育7天在含胎牛血清和无血清的培养基中培养细胞，并通过瑞氏染色分析形态学，流式细胞术分析表型，结果在含血清或无血清培养基中添加GM-CSF和IL-4后，细胞生长均产生相似的异质性群体，其中6%至10%的细胞具有同种异体刺激功能。（具有薄突起的大细胞）、免疫表型（包括CD 83+）和成熟DC的功能。肿瘤坏死因子-α显着增加了这些成熟的DC的数量，而CD 14 + PBMC的预培养耗尽几乎消除了themes.ConclusionsGeneration成熟DC在作者的无血清临床适用条件下是类似于含血清的条件，并需要CD 14+前体，分化通过CD 14-CD 83-未成熟阶段的影响下，GM-CSF和IL-4，并在TNF-α的影响下成熟为CD 83 + DC。
ObjectiveThe purpose of the study was to characterize the requirements in terms of precursors, developmental pathways, and media for the generation of large numbers of mature dendritic cells (DC) under conditions acceptable for use in adjuvant, active immunotherapy strategies for surgically treated malignancies.Summary Background DataAlthough limited previously by the small numbers accessible, DC-based immunotherapies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripheral blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generating functionally equivalent DC in serum-free conditions.MethodsPlastic-adherent PBMC (from healthy donors and patients with cancer) were incubated for 7 days with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis factor-alpha (TNF-alpha) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytometry for phenotype, and mixed lymphocyte reaction for allostimulatory function.ResultsGrowth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous population of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and function of mature DC. Tumor necrosis factor-ct significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them.ConclusionsGeneration of mature DC in the authors' serum-free clinically applicable conditions is similar to serum-containing conditions and requires CD14+ precursors, differentiation through a CD14-CD83- immature stage under the influence of GM-CSF and IL-4, and maturation into a CD83+ DC under the influence of TNF-alpha.