Differential gene expression of T-cell lymphomas (TCL) upon engagement of T-cell receptor (TCR) signaling [TCL1; T8ML1]. Homo sapiens

To study differential gene expression of TCLs upon TCR signaling, three TCL primary cells and one TCL cell line T8ML1 were chosen for this study. The prmiary TCL cells consist of TCL1 and TCL2 (sezary patients) and TCL3 (PTCL, NOS patient). The TCR signaling was engaged by anti-CD3/CD28 treatment in vitro. The TCL cells were treated without/with anti-CD3/CD28 for different time periods in vitro in cell culture. The total RNA was isolated from the TCLs and subjected to affimetry microarray (GPL17692) analysis. The differential gene expression of individual TCL was identified as well as a set of common genes invloved in TCR signaling in TCLs. The subject for this study includes pirmary cells isolated from three TCL patients [two sezary patients (TCL1 & TCL2) and one PTCL, NOS patient (TCL3)] and cells from a TCL cell line (T8ML1). The purity of isolated primary cells is >93% as idnetified by flow cytometry. The TCLs were treated with/without anti-CD3/CD28 for different time points, with 2-3 replicates for each time point. Gene expression value was presented as Log2 transformed. Differntial expressed genes were identified by ∆log2x>1 in comparison to no anti-CD3/CD28 treatment. Overall design: TCR signaling of TCLs was stimulated by anti-CD3/CD28 for vairous time points, with triplicates for each time points. The total RNAs were extracted and converted to cDNA for microarray analysis.

本研究旨在探究T细胞淋巴瘤（TCL, T-Cell Lymphoma）在T细胞受体（TCR, T-Cell Receptor）信号激活后的差异基因表达特征。本研究选取3株原发性TCL细胞与1株TCL细胞系T8ML1作为实验材料：原发性TCL细胞分别来自2名塞扎里综合征（Sezary syndrome）患者（对应TCL1、TCL2）与1例外周T细胞淋巴瘤非特指型（PTCL, NOS, Peripheral T-Cell Lymphoma, Not Otherwise Specified）患者（对应TCL3）。
通过体外添加抗CD3/CD28抗体（anti-CD3/CD28）处理，激活TCL细胞的TCR信号通路；将TCL细胞分为未处理组与抗CD3/CD28处理组，在体外细胞培养体系中设置不同的处理时长。从各组TCL细胞中提取总RNA，采用Affymetrix微阵列（GPL17692平台）进行基因表达分析。本研究不仅鉴定了单株TCL的差异基因表达谱，还筛选出了TCL中参与TCR信号通路的共有基因集。
本研究的实验对象涵盖：从3名TCL患者体内分离的原发性细胞（2名塞扎里综合征患者TCL1、TCL2与1例外周T细胞淋巴瘤非特指型患者TCL3），以及TCL细胞系T8ML1。经流式细胞术（flow cytometry）鉴定，分离得到的原发性细胞纯度均高于93%。
实验中，TCL细胞分别经抗CD3/CD28抗体处理与未处理，并设置多个时间点，每个时间点设置2-3个生物学重复。基因表达值以对数2转换（Log2 transformed）后的数值进行呈现；以与未处理组相比∆log2x>1作为筛选阈值，鉴定得到差异表达基因（differential expressed genes, DEGs）。
整体实验设计如下：通过抗CD3/CD28抗体刺激TCL的TCR信号通路，设置多个刺激时长，每个时间点设置3个生物学重复；提取各组细胞总RNA并反转录为cDNA，随后进行微阵列基因表达分析。