Comparative Analysis of Methods to Reduce Activation Signature Gene Expression in PBMCs

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with and without TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation maintained experimental flexibility while similarly down-regulating ASG expression compared to TTis. Notably, addition of TTis during cold isolation offered minimal additional advantages. Thus, while both TTis and cold isolation effectively reduced ASG expression, we found cold isolation to be a more practical approach. To study activation signature gene mitigating protocols for blood mononucleated cells (PBMCs), we isolated and treated PBMCs in different experimental conditions. Gene expression profiling was performed from patient-derived peripheral blood mononucleated cells (PBMCs) to compare two activation signature gene-mitigating cell isolation protocols; addition of transcription and translation inhibitors or cold temperature. Transcriptomic signatures were also compared from time-point thaw samples from PBMCs isolation using a standard protocol at room temperature and incubated in vitro for 0, 2, or 24 hours.

精准的细胞转录组分析离不开体内（in vivo）细胞转录组的完整保留，然而细胞分离过程中的离体（ex vivo）时长、温度等因素会诱导产生人工伪迹的基因表达，在应激响应性免疫细胞中这一现象尤为显著。本研究针对外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），探索了两种可减轻离体激活特征基因（activation signature gene, ASG）表达的策略：添加转录翻译抑制剂（transcription and translation inhibitors, TTis），以及分离过程中采用低温环境。对添加与未添加TTis分离得到的PBMCs开展对比分析，结果显示ASG的表达水平显著降低，但TTis处理会削弱后续体外实验中细胞对脂多糖（lipopolysaccharide, LPS）刺激的应答能力。与之相比，低温分离法在实现与TTis相当的ASG表达下调效果的同时，还保留了更优的实验灵活性；值得注意的是，在低温分离过程中额外添加TTis仅能带来极有限的额外益处。综上，尽管TTis与低温分离均能有效降低ASG表达，本研究认为低温分离法是更为实用的策略。为探究针对PBMCs的ASG缓解方案，我们在不同实验条件下对PBMCs进行分离与处理。本研究对患者来源的PBMCs开展基因表达谱分析，以对比两种ASG缓解型细胞分离方案：添加转录翻译抑制剂，或采用低温分离环境。此外，我们还对比了采用室温标准流程分离PBMCs，并在体外分别孵育0、2、24小时后获取的时间梯度解冻样本的转录组特征。