Role of Blimp-1 in programing Th effector cells into IL-10 producers. Mus musculus

Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10. Overall design: Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions. For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads in 96-well flat bottom plates. MACSi Beads were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to separate IL-10-secreting and non-secreting cells. Using a BD Aria or DIVA cell sorter (Becton Dickinson), living CD4+ IL-10-secreting and non-secreting cells, with (co-culture with Dll4; 'TH1Notch') or without ('TH1Control') activation of the Notch signaling pathway, were isolated. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The preparation for the hybridization to the chip was done using the GeneChip 3' IVT Express Kit. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 4 mouse genome 430 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.1.1., both Affymetrix. The data was analyzed using the original GCOS CHP-file Signals, Excel and AmiGO website.本研究对经Notch配体Delta样4（Dll4）刺激或未刺激的分泌白细胞介素10（IL-10）与不分泌IL-10的小鼠辅助性T细胞1型（Th1）开展基因表达谱分析，旨在鉴定与IL-10共表达的转录因子。 实验整体设计如下：从C57BL/6野生型小鼠的淋巴结与脾脏中分离原代初始T辅助细胞，使用美天旎生物（Miltenyi Biotec）的Multisort分选试剂盒富集CD25⁻CD4⁺CD62L⁺细胞，随后在Th1极化培养条件下进行培养。激活步骤为：将0.25×10⁶个初始T细胞与0.75×10⁶个MACSi微珠置于96孔平底培养板中共培养；接种前，MACSi微珠已用抗CD3抗体与抗CD28抗体包被（每1.0×10⁸个微珠使用30μg总原发性IgG抗体）。通过额外与共价结合重组小鼠Dll4的MACSi微珠共培养，可诱导基于Dll4的Notch信号激活。 培养5天后，采用佛波醇肉豆蔻酸乙酸酯（PMA）/离子霉素对细胞进行再刺激，并通过美天旎生物的IL-10分泌检测试剂盒分离分泌IL-10与不分泌IL-10的细胞。使用BD Aria或DIVA细胞分选仪（Becton Dickinson），分别分离存活的CD4⁺IL-10分泌型与非分泌型细胞：其中一组经Dll4共培养激活Notch信号通路，命名为‘TH1Notch’组；另一组未激活Notch信号通路，命名为‘TH1Control’组。 采用RNeasy Mini试剂盒（Qiagen）提取总RNA，通过Agilent 2100生物分析仪（Agilent，德国瓦尔德布伦）与NanoDrop ND-1000分光光度计（NanoDrop Technologies，美国特拉华州威尔明顿）对每份样品的RNA完整性与浓度进行评估。使用GeneChip 3' IVT Express试剂盒完成芯片杂交前的样本制备：将每份样品的15μg片段化互补RNA（cRNA）与共计4张小鼠基因组430 2.0基因芯片（Affymetrix）进行杂交。杂交过程在Hybridization Oven 640杂交炉中完成，随后在Fluidics Station 400流体工作站（均为Affymetrix产品）中对芯片进行洗涤与染色。最终使用GeneChip Scanner 3000扫描仪结合GCOS软件（版本1.1.1，均为Affymetrix产品）扫描芯片。 数据分析采用原始GCOS CHP文件信号值、Excel软件及AmiGO在线数据库完成。