Human memory T cells of the bone marrow are resting and maintain long-lasting memory. Homo sapiens

To compare human memory CD4+ T cell subsets in peripheral blood (PB) and bone marrow (BM) of healthy individuals at transcriptional level, we analyzed the global gene expression of ex vivo PB CD69- as well as BM CD69- and CD69+ memory CD4+ T cells from 4 paired PB and BM samples. The gene expression of these subsets was additionally compared to the transcriptional profile of 8 PB samples taken ex vivo or stimulated with phorbol myristate acetate (PMA) and Ionomycin for 3 hours. Overall design: Three ex vivo memory CD4+ T cell subsets (CD4+CD45RO+CD69+ and CD4+CD45RO+CD69- cells from bone marrow; CD4+CD45RO+CD69- cells from peripheral blood) were isolated from 4 paired bone marrow and blood samples (two males and two females) or from a different cohort of 8 blood samples (3 males and five females). The purity of sorted cells was higher than 95% as assessed by FACS. Subsequently, a fraction of purified cells from the 8 blood samples were stimulated with phorbol-myristic-acid/ionomycin (PMA/iono) for 3 h and used as high controls. Total RNA of each cell subset was extracted using a NucleoSpin RNA XS Kit (Macherey-Nagel) or 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). Double-stranded complementary RNA was synthesized from 1 µg total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to 28 HG-U133A plus 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 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

为在转录组层面比较健康个体外周血（peripheral blood, PB）与骨髓（bone marrow, BM）中的记忆性CD4+ T细胞亚群，我们分析了4对匹配的外周血与骨髓样本中离体培养的外周血CD69-、骨髓CD69-以及骨髓CD69+记忆性CD4+ T细胞的全局基因表达情况。此外，我们还将这些亚群的基因表达与8份离体或经佛波醇肉豆蔻酸乙酸酯（phorbol myristate acetate, PMA）和离子霉素（Ionomycin）刺激3小时的外周血样本的转录谱进行了比对。

总体实验设计：从4对匹配的骨髓与血液样本（2男2女），或另一组共8份血液样本（3男5女）中分离得到3种离体记忆性CD4+ T细胞亚群：骨髓来源的CD4+CD45RO+CD69+、CD4+CD45RO+CD69-细胞，以及外周血来源的CD4+CD45RO+CD69-细胞。经荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）检测，分选细胞的纯度均高于95%。随后，将8份血液样本中获得的部分纯化细胞用佛波醇肉豆蔻酸/离子霉素（PMA/iono）刺激3小时，作为高表达对照。

总RNA提取：使用NucleoSpin RNA XS试剂盒（Macherey-Nagel）或RNeasy Mini试剂盒（Qiagen）提取各细胞亚群的总RNA。通过安捷伦2100生物分析仪（Agilent，德国瓦尔德布龙）与NanoDrop ND-1000分光光度计（NanoDrop Technologies，美国特拉华州威尔明顿）对每份样本的RNA完整性与含量进行检测。

芯片杂交与扫描：从1 μg总RNA中通过Message AmpII Biotin试剂盒（Ambion，美国）合成双链互补RNA。将每份样本的15 μg片段化互补RNA与28张HG-U133A plus 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，芯片清洗与染色均在Fluidics Station 400流体工作站中进行（以上设备均为Affymetrix产品）。最后使用GeneChip Scanner 3000扫描仪搭配GCOS软件v1.4（均为Affymetrix产品）扫描芯片。

数据分析：所有经质量校验的芯片的GCOS相关数据均通过高性能芯片数据分析工具（High Performance Chip Data Analysis, HPCDA，未发表）结合BioRetis数据库（www.bioretis-analysis.de）进行分析，具体方法与验证流程已在既往研究中发表。