Expression data from immature pDC and pDC activated with CpG 1826 and influenza virus PR8. Mus musculus

CpG 1826 binds to Toll-like receptor (TLR)9, whereas influenza virus PR8 activates pDC via TLR7. Differential stimulation of pDCs is expected to result in unique activation mechanism(s) leading to a different phenotypically and functionally matured pDC We used microarrays to detail the global programme of gene expression underlying the maturation process of pDC activated with CpG 1826 and influenza virus PR8. We identified a distinct expression profile of upregulated immunomediators. Keywords: time course Overall design: Sorted pDCs were cultured for 1h and 4hs in medium control or with 5 µg/ml CpG 1826 or 300 HAU/ml purified influenza A/PR/8 virus. The first experiment (e1) included pDC in media and stimulated with CpG for 4h. In two other independent experimental batches (e2 and e3), we obtained samples of sorted pDC cultured in medium alone (med), and with CpG or PR8 (flu) for 1h and 4h. RNA extraction was performed using the RNeasy Kit (Qiagen) and hybridization on Affymetrix microarrays was performed using standard protocols. We sought to obtain homogeneous populations of pDCs at different time points under defined activation conditions in order to decipher the temporal resolution of expression profiles during the process of their maturation.

CpG 1826可结合Toll样受体9（Toll-like receptor 9, TLR9），而流感病毒PR8可通过TLR7激活浆细胞样树突状细胞（plasmacytoid dendritic cell, pDC）。对pDC的差异化刺激有望催生独特的激活机制，最终产生表型与功能均存在差异的成熟pDC群体。我们采用基因芯片（microarrays）详细解析了经CpG 1826与流感病毒PR8激活的pDC成熟过程背后的全局基因表达程序，并鉴定出了一类独特的上调免疫介导因子表达谱。关键词：时间进程（time course）。
整体实验设计：分选得到的pDC分别在仅含培养基的对照组中，或经5 μg/ml CpG 1826、300 HAU/ml纯化甲型流感病毒A/PR/8刺激下培养1小时与4小时。首个实验（e1）设置了培养基培养的pDC对照组，以及经CpG 1826刺激4小时的pDC实验组。在另外两个独立实验批次（e2与e3）中，我们获取了仅培养基培养（med）、经CpG 1826或PR8（即流感病毒组，flu）刺激1小时与4小时的分选pDC样本。RNA提取采用RNeasy试剂盒（Qiagen），在Affymetrix基因芯片上的杂交实验均遵循标准操作流程。本研究旨在获取不同激活条件下、不同时间点的均一化pDC群体，以解析其成熟过程中基因表达谱的时间动态变化规律。