Zhang Mingce, Ma Zhengyu, Selliah Nithianandan, Weiss Greta, Genin Anna, Finkel Terri H, Cron Randy Q
Division of Pediatric Rheumatology, University of Alabama at Birmingham, 1825 University Blvd,. Shelby Building, Rm. 371, Birmingham, AL 35233, United States.
Nemours/A. I. duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19803, United States.
J Immunol Methods. 2014 Jun;408:123-31. doi: 10.1016/j.jim.2014.05.014. Epub 2014 Jun 5.
Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above.
将基因导入原代人CD4 T淋巴细胞是研究T细胞依赖性免疫反应机制和人类免疫缺陷病毒1型（HIV-1）感染的关键工具。Nucleofection®是一种电穿孔技术，可实现高效的基因导入原代人CD4 T细胞，而这些细胞对传统电穿孔具有很强的抗性。尽管它在免疫学研究中很受欢迎，但尚未有文献详细描述其对CD4 T细胞生理学影响的特征。在此，我们使用新鲜分离的原代人CD4 T细胞，研究了Nucleofection®对CD4 T细胞形态、细胞内钙水平、细胞表面活化标志物和转录活性的影响。我们发现，在Nucleofection®后立即，CD4 T细胞会发生显著的形态变化，其特征是质膜起皱和扩张，1小时后恢复。细胞内钙水平在Nucleofection®后也会升高，在转染后1小时达到峰值，然后在8小时后恢复。此外，Nucleofection®导致T细胞活化标志物CD154和CD69的表达增加超过24小时，并增强了植物血凝素（PHA）刺激的活化效果。此外，即使在没有外源性刺激的情况下，转录活性在Nucleofection®后的前24小时也会增加。因此，Nucleofection®显著改变了原代人CD4 T细胞的活化状态。通过Nucleofection®导入的基因产物对CD4 T细胞功能的影响应在转染后足够的静息时间后进行评估，或根据上述活化注意事项进行分析。
