Wang Kehui, Nishimoto Kevin P, Mehta Rita S, Nelson Edward L
Department of Medicine, Division of Hematology/Oncology, School of Medicine, University of California, Irvine, USA.
J Transl Med. 2006 Apr 24;4:18. doi: 10.1186/1479-5876-4-18.
Enumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool.
Standard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cell-sorted populations from post GM-CSF samples.
Use of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Both cocktails yielded highly reproducible data. Use of the alternate cocktail: 1) yielded a putative DC population, post GM-CSF that was more homogenous and consistent with DCs, 2) resulted in less data variation across gating strategies, and 3) resulted in more uniform and concordant longitudinal data, consistent with established GM-CSF biological activity.
An alternative lineage negative cocktail substituting anti-CD66 antibody for anti-CD14 is a viable option for enumerating the circulating DC population, potentially more accurately defining the circulating DC pool by including CD14 positive immature DCs, and thus, may give more reliable data, particularly in the setting of sustained GM-CSF administration.
循环外周血树突状细胞（DCs）的计数较为复杂，原因在于缺乏一种在所有DC亚群上均表达的独特细胞表面标志物，以及使用了各种生物佐剂来调节DC区室，包括粒细胞巨噬细胞集落刺激因子（GM-CSF）。常用方法采用抗体组合，通常包括抗CD14，以定义一个谱系阴性、MHC II类阳性的假定DC群体。报道的流式细胞术方案包括高度可变的设门策略和DC识别标准。对DC多形性、GM-CSF生物学的认识不断增加，以及对某些DC亚群中CD14表达的认识，促使我们考虑使用另一种谱系组合来改进对循环DC池的识别。
在接受每日GM-CSF造血生长因子支持的剂量密集型细胞毒性化疗前后，从正常供体和乳腺癌患者获取的样本上，用与MHC II类和包含标准CD14或包含替代CD66acde的谱系组合的适当荧光染料偶联抗体进行标准全血染色。通过标准流式细胞术对假定的DC进行计数。使用双尾Mann-Whitney或Wilcoxon符号秩检验评估数据集差异。在GM-CSF处理后的样本的细胞分选群体中检查细胞形态。
在正常供体和癌症患者基线时，使用任何一种抗体组合都能定义出大小相当的谱系阴性、MHC II类阳性群体。然而，选择MHC II类表达水平不断增加的谱系阴性亚群，使用替代组合可鉴定出更大的假定DC群体。两种组合都产生了高度可重复的数据。使用替代组合：1）产生了一个假定的DC群体，GM-CSF处理后该群体更均匀且与DC一致，2）导致不同设门策略的数据变异性更小，3）产生了更均匀和一致的纵向数据，与已确立的GM-CSF生物学活性一致。
用抗CD66抗体替代抗CD14的另一种谱系阴性组合是计数循环DC群体的可行选择，通过纳入CD14阳性未成熟DC，可能更准确地定义循环DC池，因此，可能提供更可靠的数据，特别是在持续给予GM-CSF的情况下。
