Gene expression profiling of Th17, Th1*, Treg cells in the spinal cord of EAE mice , and Th17, Treg cells in the SILP of SFB colonized mice. Gene expression profiling of Th17, Th1*, Treg cells in the spinal cord of EAE mice , and Th17, Treg cells in the SILP of SFB colonized mice

The goal of the RNAseq experiment is to compare gene expression of T cells in different microenvironments. We hypothesized that same T cell subtype (Th17/Th1* cells, or Treg cells in this study) need to change gene expression to adapt to different microenvironments (spinal cord in EAE mice VS. SILP in SFB-colonized mice). By profiling and comparing gene expression of these cells, we were able to identify candidate genes that enable cells for tissue adaptation for functional study. Overall design: The mice used for T cell isolation were 10-week old IL-17aGfp/+ Foxp3Rfp male littermates (two biological replicates for each sample). Each CD4+ T cell sub-population was sorted with Aria II (EAE Th17: IL-17a+ Klrc1- Foxp3-; EAE Th1*: Klrc1+ Foxp3-; EAE Treg: Foxp3+ IL-17a- Klrc1-; SFB Th17: IL-17a+ Foxp3-; SFB Treg: IL-17a- Foxp3+). EAE samples were isolated at the peak of the disease (Day15, score 5). SFB samples were isolated from stable SFB-colonized mice.

本RNA测序（RNAseq）实验旨在对比不同微环境中T细胞的基因表达谱。本研究假设：同一T细胞亚群（辅助性T细胞17（Th17）、辅助性T细胞1*（Th1*）或本研究中的调节性T细胞（Treg））需通过改变基因表达以适应不同微环境——即实验性自身免疫性脑脊髓炎（EAE）模型小鼠的脊髓，与分段丝状菌（SFB）定植小鼠的小肠固有层（SILP）。通过对上述细胞的基因表达谱进行分析与比对，本研究得以筛选出可用于组织适应性功能研究的候选基因。

总体实验设计如下：用于分离T细胞的实验动物为10周龄的IL-17aGfp/+ Foxp3Rfp雄性同窝仔鼠，每份样本设置2个生物学重复。采用Aria II流式细胞分选仪对各CD4+ T细胞亚群进行分选：EAE模型小鼠Th17细胞：IL-17a+ Klrc1- Foxp3-；EAE模型小鼠Th1*细胞：Klrc1+ Foxp3-；EAE模型小鼠Treg细胞：Foxp3+ IL-17a- Klrc1-；SFB定植小鼠Th17细胞：IL-17a+ Foxp3-；SFB定植小鼠Treg细胞：IL-17a- Foxp3+。EAE模型样本于疾病峰值期（第15天，评分5分）分离获取；SFB定植样本则取自稳定定植SFB的小鼠。