Nguyen X D, Müller-Berghaus J, Kälsch T, Schadendorf D, Borggrefe M, Klüter H
Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, Red-Cross Blood Donation Service of Baden-Wurttemberg-Hessen, Germany.
Cytotherapy. 2008;10(7):720-9. doi: 10.1080/14653240802378912.
Monocytapheresis has been established to collect a sufficient number of monocytes (MO) for differentiation to dendritic cells (DC) as a cancer vaccine. Platelets (Plt) are invariably found as a contaminant in the final monocytapheresis product. The aim of this study was to investigate DC differentiation under the influence of Plt with regard to their function and phenotype.
MO were isolated and co-cultured with autologous Plt at different MO:Plt ratios (1:1.7, 1:5, 1:15, 1:45 and 1:135) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-12p70 release after ligation of CD40L was determined in the supernatant by enzyme-linked immunosorbent assay (ELISA). For T-cell stimulation, tetanus toxoid was added to immature DC and maturation was induced by adding cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha and prostaglandin E(2)). Stimulated T cells were analyzed for activation and proliferation as well as for intracellular cytokines by flow cytometry.
All DC cultures were strongly positive for CD83. At a contaminating concentration of 5 Plt/MO, matured DC showed the highest expression of HLA-DR, CD80 and CD86, inducing a strong T-cell proliferation with high production of IL-4 and interferon-gamma. The highest level of IL-12p70 production was observed by the same DC group.
Plt did not negatively influence DC maturation but enhanced the expression of co-stimulatory molecules and the release of IL-12. Functionally this was reflected by a strong T-cell response that involved T-helper 1 (Th1)- as well as Th2-biased T cells. Our findings show that controlling the Plt concentration may provide important advantages for the generation of DC for use in immunotherapy.
已证实单采单核细胞术可收集足够数量的单核细胞（MO）以分化为树突状细胞（DC）作为癌症疫苗。血小板（Plt）始终作为最终单采单核细胞术产品中的污染物被发现。本研究的目的是探讨在Plt影响下DC的分化及其功能和表型。
分离MO并在白细胞介素-4（IL-4）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）存在的情况下，以不同的MO:Plt比例（1:1.7、1:5、1:15、1:45和1:135）与自体Plt共培养。通过酶联免疫吸附测定（ELISA）测定CD40L连接后上清液中IL-12p70的释放。为了刺激T细胞，将破伤风类毒素添加到未成熟DC中，并通过添加细胞因子（IL-1β、IL-6、肿瘤坏死因子-α和前列腺素E2）诱导成熟。通过流式细胞术分析刺激后的T细胞的活化、增殖以及细胞内细胞因子。
所有DC培养物的CD83均呈强阳性。在污染浓度为5个Plt/MO时，成熟DC显示出最高水平的HLA-DR、CD80和CD86表达，诱导强烈的T细胞增殖并产生大量的IL-4和干扰素-γ。同一DC组观察到IL-12p70产生的最高水平。
Plt不会对DC成熟产生负面影响，反而会增强共刺激分子的表达和IL-12的释放。在功能上，这表现为涉及辅助性T细胞1（Th1）和Th2偏向性T细胞的强烈T细胞反应。我们的研究结果表明，控制Plt浓度可能为用于免疫治疗的DC的产生提供重要优势。
