Ets1 dampens expression of Tbet and CD8- or NK-like effector programing in postselection iNKT1 cells [RNA-seq]. Ets1 dampens expression of Tbet and CD8- or NK-like effector programing in postselection iNKT1 cells [RNA-seq]

The goal of this study was to compare the gene expression program of thymic and liver iNKT1 cells from Control mice to those with a postselection deletion of the transcription factor Ets1. Ets1-floxed alleles were deleted in iNKT1 cells using Tbx21Cre bacterial artifical chromosome transgenic mice that also carry a Rosa26-floxed-stop-YFP reporter. Control iNKT1 cells were Tbx21Cre+ Rosa26-floxed-stop-YFP reporter+. We examined gene expression by RNA-sequencing of sorted iNKT1 cells (CD1d-Tet-PBS57+ YFP+) from the thymus and liver. Reads were aligned to the mm10 reference genome using Tophat v 2.1.0. Reads were assigned to genes using the htseq-count tool from HTSeq v 0.6.1 and gene annotations from Ensembl release 78. Differential expression was calculated across 3 independent replicates by EdgeR. We found that iNKT1 cells lacking Ets1 upregulated Tbx21 and its protein Tbet, many Tbet target genes, and CD8 effector T cell associated genes. Overall design: Expression profiling analysis of RNA from thymic and liver iNKT1 cells isolated from Ctrl (Tbx21Cre; Rosa26-YFP+) and cKO (Tbx21Cre; Rosa26-YFP+; Ets1F/F) mice by RNA-sequencing. 3 independent replicates for each sample.

本研究旨在对比对照组小鼠与转录因子Ets1经阳性选择后缺失的小鼠的胸腺及肝脏iNKT1细胞（iNKT1 cells）的基因表达程序。本研究通过同时携带Rosa26-floxed-stop-YFP报告基因的Tbx21Cre细菌人工染色体转基因小鼠，在iNKT1细胞中敲除Ets1-floxed等位基因。对照组iNKT1细胞为Tbx21Cre+ Rosa26-floxed-stop-YFP报告基因阳性细胞。我们通过RNA测序（RNA-sequencing）对分选自胸腺与肝脏的iNKT1细胞（CD1d-Tet-PBS57+ YFP+）进行基因表达检测。使用Tophat v2.1.0将测序读段比对至mm10参考基因组。借助HTSeq v0.6.1中的htseq-count工具，并结合Ensembl release 78的基因注释信息，将测序读段分配至对应基因。通过EdgeR工具对3组独立生物学重复样本进行差异表达分析。研究结果显示，缺失Ets1的iNKT1细胞会上调Tbx21及其编码蛋白Tbet的表达、大量Tbet靶基因以及CD8效应T细胞相关基因的转录水平。实验整体设计：通过RNA测序对分离自对照组（Ctrl，Tbx21Cre；Rosa26-YFP+）与条件性敲除组（cKO，Tbx21Cre；Rosa26-YFP+；Ets1F/F）小鼠的胸腺、肝脏iNKT1细胞的RNA进行表达谱分析。每个样本设置3次独立生物学重复。